(FREE PDF Sample) Microcalorimetry of Biological Molecules Methods and Protocols Eric Ennifar Ebooks

Full download test bank at ebook textbookfull.
com
Microcalorimetry of Biological
Molecules Methods and Protocols
CLICK LINK TO DOWLOAD
https://textbookfull.com/product/microcalorim
etry-of-biological-molecules-methods-and-
protocols-eric-ennifar/
textbookfull
More products digital (pdf, epub, mobi) instant
download maybe you interests ...
Nucleic Acid Crystallography Methods and Protocols 1st

Edition Eric Ennifar (Eds.)
https://textbookfull.com/product/nucleic-acid-crystallography-
methods-and-protocols-1st-edition-eric-ennifar-eds/
Zebrafish Methods and Protocols Koichi Kawakami
https://textbookfull.com/product/zebrafish-methods-and-protocols-
koichi-kawakami/
SNAREs: Methods and Protocols Rutilio Fratti
https://textbookfull.com/product/snares-methods-and-protocols-
rutilio-fratti/
Epitranscriptomics: Methods and Protocols Narendra

Wajapeyee
https://textbookfull.com/product/epitranscriptomics-methods-and-
protocols-narendra-wajapeyee/
Phytoplasmas: Methods and Protocols Rita Musetti
https://textbookfull.com/product/phytoplasmas-methods-and-
protocols-rita-musetti/
Metalloproteins: Methods and Protocols Yilin Hu
https://textbookfull.com/product/metalloproteins-methods-and-
protocols-yilin-hu/
Nanotoxicity: Methods and Protocols Qunwei Zhang
https://textbookfull.com/product/nanotoxicity-methods-and-
protocols-qunwei-zhang/
Autoantibodies: Methods and Protocols Gunnar Houen
https://textbookfull.com/product/autoantibodies-methods-and-
protocols-gunnar-houen/
Oligodendrocytes: Methods and Protocols David Lyons
https://textbookfull.com/product/oligodendrocytes-methods-and-
protocols-david-lyons/
Methods in
Molecular Biology 1964
Eric Ennifar Editor
Microcalorimetry
of Biological
Molecules
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:

http://www.springer.com/series/7651
Microcalorimetry of Biological
Molecules
Methods and Protocols
Edited by
Eric Ennifar
Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, CNRS, Strasbourg, France
Editor
Eric Ennifar
Institut de Biologie Moléculaire et Cellulaire
Université de Strasbourg, CNRS
Strasbourg, France
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-9178-5 ISBN 978-1-4939-9179-2 (eBook)
https://doi.org/10.1007/978-1-4939-9179-2
Library of Congress Control Number: 2019935496
© Springer Science+Business Media, LLC, part of Springer Nature 2019

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,
even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations
and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to
be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.
The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
This Humana Press imprint is published by the registered company Springer Science+Business Media, LLC, part of
Springer Nature.
The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.
Preface
Although structural biology is key to elucidate molecular architecture of biological mole-

cules, thermodynamic and kinetic data are yet an essential and necessary complement to
structural studies for a comprehensive understanding of binding mechanisms and mechan-
isms of action. Structural data alone, even when associated with sophisticated computational
methods, cannot fully define the driving forces for binding interactions or even accurately
predict their binding affinities or kinetics. Thermodynamics and kinetics provide quantita-
tive data required to understand these driving forces, the time in which drug and its target
associate and dissociate, and for evaluating and understanding at a deeper level the effects of
substituent changes on binding affinity. Microcalorimetry is the only direct method yielding
thermodynamic parameters without the use of any label. Among the key advantages, it is
relatively artifacts-free, not limited by the ligand or protein sizes, and offers a precise
determination of affinities among other key data.
The first part of this volume is dedicated to differential scanning calorimetry (DSC), the
technique of choice for evaluation of protein stability, but which can also be valuable for the
study of biofluids as shown here. DSC is a quite well-established technique in the pharma-
ceutical industry but would also benefit to academic research where it is not widely available.
The second part is devoted to isothermal titration calorimetry (ITC). The use of ITC in
biology has considerably grown in the past 10 years, mostly as a consequence of the
availability of high-sensitivity microcalorimeters requiring low sample volume. The inevita-
ble side effect of this rapid dissemination is a frequent misuse of this technique, being
recurrently used to decorate publications in structural biology by providing Kd (dissociation
constant) tables. However, alternative techniques can provide similar data about affinity with
much lower sample requirements. ITC, however, can do much more and can go well beyond
Kds. ITC users and the scientific community would clearly benefit from taking full advantage
of the technique as illustrated in these chapters. The third and last part includes advanced
data processing and is intended to a more experienced audience. It will be useful for readers
dealing with complex molecular systems (most interactions are much more complex than
initially thought!) or for those who are interested into retrieving kinetic data in addition to
thermodynamic data. ITC indeed recently appears as an interesting and reliable technique to
gain insights into kinetic data, similarly to surface plasmon resonance. It is a safe bet that
these kinetic aspects of ITC are likely to grow in the future.
Finally, I would like to thank the authors who participated in the production of this
book, for their contribution and for their patience. I hope this volume will provide new
stimulating ideas for readers in using microcalorimetry for their experiments.
Strasbourg, France Eric Ennifar
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
PART I DIFFERENTIAL SCANNING CALORIMETRY

1 The Contribution of Differential Scanning Calorimetry for the Study
of Peptide/Lipid Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Marie-Lise Jobin and Isabel D. Alves
2 Protocols of IATC, DSC, and PPC: The Multistate Structural
Transition of Cytochrome c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Shigeyoshi Nakamura and Shun-ichi Kidokoro
3 Value of DSC in Characterization and Optimization of Protein Stability . . . . . . . 33
Katherine Bowers and Natalia Markova
4 Plasmatic Signature of Disease by Differential Scanning Calorimetry (DSC). . . . 45
Philipp O. Tsvetkov and François Devred
PART II ISOTHERMAL TITRATION CALORIMETRY
5 Intrinsic Thermodynamics of Protein-Ligand Binding by Isothermal

Titration Calorimetry as Aid to Drug Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Vaida Paketurytė, Asta Zubrienė, John E. Ladbury,
and Daumantas Matulis
6 Isothermal Titration Calorimetry Measurements of Riboswitch-Ligand
Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Christopher P. Jones, Grzegorz Piszczek, and Adrian R. Ferré-D’Amaré
7 ITC Studies of Ribosome/Antibiotics Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Emma Schenckbecher, Benoı̂t Meyer, and Eric Ennifar
8 High-Quality Data of Protein/Peptide Interaction by Isothermal
Titration Calorimetry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Juan Ramirez and Yves Nominé
9 ITC Measurement for High-Affinity Aptamers Binding to Their Target
Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Ryo Amano, Tomohisa Furukawa, and Taiichi Sakamoto
10 Thermodynamics of Molecular Machines Using Incremental ITC . . . . . . . . . . . . 129
Benoı̂t Meyer, Cyrielle da Veiga, Philippe Dumas, and Eric Ennifar
11 Measuring the Metabolic Activity of Mature Mycobacterial Biofilms
Using Isothermal Microcalorimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Anna Solokhina, Gernot Bonkat, and Olivier Braissant
vii
viii Contents
12 Characterization of Microtubule-Associated Proteins (MAPs)

and Tubulin Interactions by Isothermal Titration Calorimetry (ITC). . . . . . . . . . 151
Philipp O. Tsvetkov, Romain La Rocca, Soazig Malesinski,
and François Devred
PART III ADVANCED DATA PROCESSING AND KINETICS
13 Analysis of Isothermal Titration Calorimetry Data for Complex

Interactions Using I2CITC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Ibrahim Q. Saeed and Niklaas J. Buurma
14 Tinkering with Binding Polynomials in Isothermal Titration Calorimetry . . . . . . 185
Rafael Claveria-Gimeno, Sonia Vega, Olga Abian,
and Adrian Velazquez-Campoy
15 The Use of ITC and the Software AFFINImeter for the Quantification
of the Anticoagulant Pentasaccharide in Low Molecular Weight Heparin . . . . . . 215
Eva Muñoz and Juan Sabı́n
16 Thermodynamic and Kinetic Analysis of Isothermal Titration
Calorimetry Experiments by Using KinITC in AFFINImeter. . . . . . . . . . . . . . . . . 225
Eva Muñoz, Juan Sabı́n, Javier Rial, Daniel Pérez, Eric Ennifar,
Philippe Dumas, and Ángel Piñeiro
17 Enzyme Kinetics Determined by Single-Injection Isothermal Titration
Calorimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Colette F. Quinn and Lee D. Hansen
18 Characterization of Enzymatic Reactions Using ITC . . . . . . . . . . . . . . . . . . . . . . . . 251
Barbara Zambelli
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Contributors
OLGA ABIAN Institute of Biocomputation and Physics of Complex Systems (BIFI), Joint
Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Universidad de Zaragoza, Zaragoza,
Spain; Instituto Aragonés de Ciencias de la Salud (IACS), Zaragoza, Spain; Aragon
Institute for Health Research (IIS Aragon), Zaragoza, Spain; Department of Biochemistry
and Molecular and Cell Biology, Universidad de Zaragoza, Zaragoza, Spain; Centro de
Investigacion Biomédica en Red en el Área Temática de Enfermedades Hepáticas y
Digestivas (CIBERehd), Barcelona, Spain
ISABEL D. ALVES Chimie et Biologie des Membranes et Nanoobjets, CBMN CNRS UMR
5248, Université Bordeaux 1, Pessac, France
RYO AMANO Faculty of Advanced Engineering, Department of Life Science, Chiba Institute
of Technology, Narashino-shi, Chiba, Japan
GERNOT BONKAT alta uro AG, Basel, Switzerland
KATHERINE BOWERS Principal Scientist/Group Leader Analytical and Formulation
Development, FUJIFILM Diosynth Biotechnologies U.S.A., Inc., Morrisville, NC, USA
OLIVIER BRAISSANT Center of Biomechanics and Biocalorimetry, University Basel, c/o
Department of Biomedical Engineering (DBE), Allschwil, Switzerland
NIKLAAS J. BUURMA Physical Organic Chemistry Centre, School of Chemistry, Cardiff
University, Cardiff, UK
RAFAEL CLAVERIA-GIMENO Institute of Biocomputation and Physics of Complex Systems
(BIFI), Joint Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Universidad de Zaragoza,
Zaragoza, Spain; Instituto Aragonés de Ciencias de la Salud (IACS), Zaragoza, Spain;
Aragon Institute for Health Research (IIS Aragon), Zaragoza, Spain
CYRIELLE DA VEIGA Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg,
CNRS, Strasbourg, France
FRANÇOIS DEVRED Aix-Marseille Univ, CNRS, INP, Inst Neurophysiopathol, Fac Pharm,
Marseille, France
PHILIPPE DUMAS Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg,
ERIC ENNIFAR Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg,
ADRIAN R. FERRÉ-D’AMARÉ Biochemistry and Biophysics Center, National Heart, Lung
and Blood Institute, National Institutes of Health, Bethesda, MD, USA
TOMOHISA FURUKAWA Faculty of Advanced Engineering, Department of Life Science, Chiba
Institute of Technology, Narashino-shi, Chiba, Japan
LEE D. HANSEN Department of Chemistry and Biochemistry, Brigham Young University,
Provo, UT, USA
MARIE-LISE JOBIN Institute for Pharmacology and Toxicology, Rudolf Virchow Center—Bio-
Imaging Center, University of Würzburg, Würzburg, Germany
CHRISTOPHER P. JONES Biochemistry and Biophysics Center, National Heart, Lung and
Blood Institute, National Institutes of Health, Bethesda, MD, USA
SHUN-ICHI KIDOKORO Department of Bioengineering, Nagaoka University of Technology,
Nagaoka, Japan
ix
x Contributors
JOHN E. LADBURY Department of Molecular and Cell Biology and Astbury Centre for
Structural Biology, University of Leeds, Leeds, UK
ROMAIN LA ROCCA Aix-Marseille Univ, CNRS, INP, Inst Neurophysiopathol, Fac Pharm,
Marseille, France
SOAZIG MALESINSKI Aix-Marseille Univ, CNRS, INP, Inst Neurophysiopathol, Fac Pharm,
Marseille, France
NATALIA MARKOVA MicroCal, Malvern Panalytical, Uppsala, Sweden
DAUMANTAS MATULIS Department of Biothermodynamics and Drug Design, Institute of
Biotechnology, Vilnius University, Vilnius, Lithuania
BENOÎT MEYER Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg,
EVA MUÑOZ AFFINImeter Scientific & Development Team, Software 4 Science
Developments, S. L. Ed. Emprendia, Santiago de Compostela, A Coruña, Spain
SHIGEYOSHI NAKAMURA Department of General Education, National Institute of
Technology, Ube College, Ube, Japan
YVES NOMINÉ Equipe Labelisée Ligue 2015, Department of Integrative Structural Biology,
Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964
UMR 7104 CNRS, Université de Strasbourg, Illkirch, France
VAIDA PAKETURYTĖ Department of Biothermodynamics and Drug Design, Institute of
DANIEL PÉREZ AFFINImeter Scientific & Development Team, Software 4 Science
ÁNGEL PIÑEIRO AFFINImeter Scientific & Development Team, Software 4 Science
Developments, S. L. Ed. Emprendia, Santiago de Compostela, A Coruña, Spain; Soft
Matter & Molecular Biophysics Group, Departamento de Fı́sica Aplicada, Facultad de
Fı́sica, Universidade de Santiago de Compostela, Santiago de Compostela, Spain
GRZEGORZ PISZCZEK Biophysics Core Facility, National Heart, Lung and Blood Institute,
National Institutes of Health, Bethesda, MD, USA
COLETTE F. QUINN Applications Lab, TA Instruments, Lindon, UT, USA
JUAN RAMIREZ Institute of Nanotechnology (INT), Karlsruhe Institute of Technology (KIT),
Eggenstein-Leopoldshafen, Germany
JAVIER RIAL AFFINImeter Scientific & Development Team, Software 4 Science
JUAN SABÍN AFFINImeter Scientific & Development Team, Software 4 Science
IBRAHIM Q. SAEED Department of Chemistry, College of Science, Salahaddin University,
Erbil, Kurdistan Region, Iraq
TAIICHI SAKAMOTO Faculty of Advanced Engineering, Department of Life Science, Chiba
Institute of Technology, Narashino-shi, Chiba, Japan
EMMA SCHENCKBECHER Institut de Biologie Moléculaire et Cellulaire, Université de
Strasbourg, CNRS, Strasbourg, France
ANNA SOLOKHINA Center of Biomechanics and Biocalorimetry, University Basel, c/o
Department of Biomedical Engineering (DBE), Allschwil, Switzerland
PHILIPP O. TSVETKOV Aix-Marseille Univ, CNRS, INP, Inst Neurophysiopathol,
Fac Pharm, Marseille, France
SONIA VEGA Institute of Biocomputation and Physics of Complex Systems (BIFI), Joint Units
IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Universidad de Zaragoza, Zaragoza, Spain
Contributors xi
ADRIAN VELAZQUEZ-CAMPOY Institute of Biocomputation and Physics of Complex Systems

(BIFI), Joint Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Universidad de Zaragoza,
Zaragoza, Spain; Aragon Institute for Health Research (IIS Aragon), Zaragoza, Spain;
Department of Biochemistry and Molecular and Cell Biology, Universidad de Zaragoza,
Zaragoza, Spain; Centro de Investigacion Biomédica en Red en el Área Temática de
Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain; Fundacion ARAID,
Government of Aragon, Zaragoza, Spain
BARBARA ZAMBELLI Laboratory of Bioinorganic Chemistry, Department of Pharmacy and
Biotechnology, University of Bologna, Bologna, Italy
ASTA ZUBRIENĖ Department of Biothermodynamics and Drug Design, Institute of
Part I
Differential Scanning Calorimetry

Chapter 1
The Contribution of Differential Scanning Calorimetry

for the Study of Peptide/Lipid Interactions
Marie-Lise Jobin and Isabel D. Alves
Abstract
Membrane-active peptides include a variety of molecules such as antimicrobial (AMP), cell-penetrating
(CPP), viral, and amyloid peptides that are implicated in several pathologies. They constitute important
targets because they are either at the basis of novel therapies (drug delivery for CPPs or antimicrobial
activity for AMPs) or they are the agents causing these pathologies (viral and amyloid peptides). They all
share the common property of interacting with the cellular lipid membrane in their mode of action.
Therefore, a better understanding of the peptide/lipid (P/L) interaction is essential to help decipher
their mechanism of action. Among the different biophysical methods that can be used to fully characterize
P/L interactions, differential scanning calorimetry (DSC) allows determining the peptide effect on the lipid
phase transitions, a property that reflects the P/L interaction mode. A general protocol for classical DSC
experiments for P/L studies will be provided.
Key words Membrane-active peptides, Peptide/lipid interaction, Differential scanning calorimetry,

Lipid phase transition, Thermodynamic behavior
1 Introduction
Membrane lipid properties (fluidity, thickness, ordering, etc.) vary

with temperature. The lipid membrane undergoes transition states
as the temperature increases, and the temperatures at which those
occur are called phase transition temperatures. Lipid phase transi-
tions are totally reversible. As the hydrocarbon length is increased,
van der Waals interactions become stronger requiring more energy
to disrupt the ordered packing and arising to higher transition
phase temperatures. When the number of lipid acyl chain unsatura-
tion increases, the transition temperature decreases due to a disrup-
tion of acyl chain packing.
For lipids forming lamellar phases, at low temperatures, lipids
are in the gel phase (Lβ0 ), a rigid phase where lipids are highly
ordered and closely packed. As the temperature increases, the tilt-
ing of lipid headgroups changes, and lipids enter the rippled phase
Eric Ennifar (ed.), Microcalorimetry of Biological Molecules: Methods and Protocols, Methods in Molecular Biology, vol. 1964,
https://doi.org/10.1007/978-1-4939-9179-2_1, © Springer Science+Business Media, LLC, part of Springer Nature 2019
3
4 Marie-Lise Jobin and Isabel D. Alves
Fig. 1 Thermogram of a lamellar phase transition showing the three typical lipid phases (gel phase Lβ0 , rippled
phase Pβ0 , and liquid phase Lα) and the corresponding temperature transitions (Tpre and Tm)
(Pβ0 ). In this intermediate phase, the bilayer is still highly ordered

with low degree of fluidity and has a series of ripples along the
surface. The phase transition associated is called the pre-transition,
and the temperature at which it occurs is the pre-transition temper-
ature (Tpre) (Fig. 1). The pre-transition is often only visible for
single-component lipid vesicles and tends to be absent when a
mixture of lipids is studied. At higher temperatures, lipids undergo
the main phase transition to a liquid-disordered phase (Lα) that is
accompanied by an increase in lipid disordering, decrease in bilayer
thickness, and lateral expansion [1]. The main transition is due to a
cooperative melting of the hydrocarbon changes. It includes a
conformational change of the hydrocarbon chains from all trans
in the rigid gel state to a disordered state that allows gauche con-
formations. Accompanying these changes are a lateral expansion
due to increased lipid mobility and a decrease in bilayer thickness.
The main transition occurs at a higher temperature and is faster and
associated with a larger enthalpy change than the pre-transition.
Lipid phase transition properties are modulated by lipid physi-
cochemical properties as fatty acid chain length, degree of unsatura-
tion, and nature and size of their polar headgroup and by the buffer
conditions (ionic strength, type of ions, pH) and external factors as
pressure [2–4].
The lamellar transitions are the most studied since they are the
most common in biological systems as they are adopted, among
others, by phosphatidylcholines (PC), the most abundant phos-
pholipid in eukaryotic cell membranes. An exception are lipids
that form hexagonal phases such as the phosphatidylethanolamines
which, besides the lamellar phase, possess an additional phase tran-
sition between lamellar (Lα) and hexagonal phase (HII, also called
inverted micelle). The temperature phase transition between these
DSC Studies of P/L Interactions 5
two phases is called TH and occurs at higher temperatures than Tm

[2, 5–7].
One of the biophysical approaches that allow a full characteri-
zation of lipid phase transitions is differential scanning calorimetry
(DSC). DSC can measure phase transition temperatures (Tpre, Tm,
TH) as well as enthalpy (ΔH) and cooperativity of the phase transi-
tions by measuring the difference of temperature needed to com-
pensate between the thermodynamic behaviors of two cells: one
“inert” reference cell filled with aqueous buffer and one sample cell
filled with the sample to be analyzed [5, 8, 9]. In a DSC measure-
ment, the difference in the amount of heat required to increase the
temperature of a sample and reference are measured as a function of
temperature. The basic principle underlying this technique is that,
when the sample undergoes a physical transformation such as phase
transitions, more or less heat will need to flow to it than the
reference to maintain both at the same temperature. Whether less
or more heat must flow to the sample depends on whether the
process is exothermic or endothermic. By observing the difference
in heat flow between the sample and reference, differential scanning
calorimeters are able to measure the amount of heat absorbed or
released during such transitions. This heat is then converted in
specific heat capacity (Cp that is expressed in calories or joules per
mol per C) that is usually employed in data presentation.
Three parameters of interest can be extracted from DSC ther-
mograms: the area under the transition peak, which is proportional
to the enthalpy of the transition (ΔH) and directly correlates to the
strength of van der Walls forces between the lipid fatty acid chains,
the full width at half maximum (FWHM) of the transition peak
(ΔT1/2) that reflects the cooperativity of the transition related with
the number of molecules that undergo a transition simultaneously,
and the transition temperature itself (Tpre, Tm, TH).
DSC can be used to monitor phase transitions of single, binary,
or simple lipid mixtures allowing to better mimic more physiologi-
cal membrane systems. One should keep in mind that for lipid
samples composed of more than one lipid component, the lipids
must be appropriately chosen to ensure proper miscibility (for
reports on lipid miscibility, see refs. 10–14). Additionally certain
lipids such as cholesterol have a strong impact in the phase transi-
tion due to their natural intercalation between the fatty acid chains.
Cholesterol broadens the endotherm and can even completely
abolish the transition [15, 16]; therefore, their content must be
kept low. At concentrations below 10%, cholesterol induces mini-
mal phase separation. Lipid miscibility will be further discussed
below.
The mode of interaction of peptides with lipids can be accu-
rately studied using DSC since this method is nonintrusive and
doesn’t implicate the use of any probe. As peptides interact with
lipids, they can induce changes in lipid properties as lipid packing,
membrane fluidity, cooperativity of the transition, and others that

are reflected in the different parameters measured by the DSC
experiment. Very generally, the pre-transition is very sensitive to
all molecules that establish contacts with the lipid headgroups and
tends to disappear when peptides are added. On the other hand,
molecules that affect the main phase transition act at a deeper level
than those that only affect the pre-transition so with insertion
below the headgroup level [5, 17].
2 Materials
1. Lipids for the studies presented here were obtained from the
Avanti Polar Lipids (Alabama, USA).
2. Buffer for DSC studies: 10 mM Tris–HCl, 0.1 M NaCl, 2 mM
EDTA, pH 7.6. Other buffers can be used considering that
they do not undergo heat changes for the temperature ranges
used in the measurements (see Note 1).
3. Peptides were purchased from companies or synthesized by
classical solid-phase peptide chemistry procedures using Fmoc
strategy, purified to 90% purity and lyophilized to a powder.
4. DSC scans were obtained with a Nano DSC (TA Instruments)
equipped with U-shape platinum cells. Other DSC instruments
can be used.
3 Methods
3.1 The Lipid Model The most commonly used lipid model system used for DSC studies
System are multilamellar vesicles (MLVs) because these systems are very
easy to prepare and are the most stable lipid model systems
[18]. Moreover, MLVs also result in a more homogeneous sample
and in a higher and better resolved DSC signal than other types of
liposomes as small and large unilamellar vesicles [5, 19]. Regarding
the composition of the lipid vesicles, one needs to choose lipids that
are the most representatives of the in vivo system that one wants to
mimic but needs to keep in mind the limitations associated with the
method. Some additional considerations about the type and com-
position of the lipid model system are provided in Notes 2 and 3.
1. A lipid film is prepared by dissolving the chosen lipid (1 mg
when a single lipid is used and to investigate gel to fluid phase
transition; for other conditions see Note 3) in minimal amount
of chloroform and methanol (only if not soluble in chloroform
alone, as it occurs often with anionic lipids) necessary to allow
complete dissolution. For lipid mixtures, it is very important
that lipids are thoroughly mixed and dissolved. To this aim the
proportion of chloroform and methanol can be adjusted

depending on the lipid to be dissolved. For example, if choles-
terol is present, the methanol content should be reduced to
avoid crystallization of cholesterol. The same is valid for mix-
tures containing phosphatidylethanolamine. The film is evapo-
rated under a flow of nitrogen and then subjected to total
evaporation under high vacuum to remove all traces of organic
solvent (2 h, depending on the system power).
2. The lipid film is hydrated by adding 1 mL of the chosen buffer
system (see Note 1), and then MLVs are spontaneously formed
by strong vortex. For lipids with a lipid phase transition tem-
perature (Tm) that is considerably above room temperature, the
sample should be warmed to a temperature above Tm to allow
complete hydration and proper formation of MLVs.
3. If unilamellar lipid vesicles (such as large ones, LUVs) are used,
then after step 2, the MLVs are subjected to six steps of totally
freezing/thawing (by immersing samples in liquid nitrogen
followed by sample heating in a water bath at temperatures
above the lipid phase transition). After that, the liposomes size
is homogenized by passing the solution through a mini
extruder equipped with 100 nm pore filter (Whatman). Due
to a slight loss of lipid during the extrusion process (~5%), the
lipid concentration should be determined by the Rouser
method that can accurately quantify phosphate content in
samples [20].
3.2 The DSC The DSC instrument is composed of two cells, a reference and a
Measurement sample cell. In the reference cell, the exact same buffer used for the
sample (blank) must be loaded. In the sample cell, the lipid solution
or the lipid and peptide mixture is loaded. The volume of the cells
varies from one instrument to another (the studies reported here
were performed with a DSC with 300 μL cells).
1. Blank measurement should be performed first. For that the
same buffer should be loaded in both the reference and the
cell sample. Before loading, the buffer solution should be
degassed for about 15 min to remove possible air bubbles.
The same applies for all samples to be loaded in the DSC
cells. Air bubbles displace liquid and therefore reduce the
heat capacity (yielding erroneous results). As air bubbles can
dissolve into solution over time, this will result in aberrant
increase in heat capacity each subsequent cycle as the bubble
dissolves. After loading the DSC cell, the fact that the cell is
kept under pressure (~35 psi) minimizes air bubbles. The
temperature scan measurement should be performed over a
large temperature range (to include the temperature range
that is used for samples to be measured) and at a rate of
1 C/min with a delay of 10–15 min between sequential scans

in a series that allows for thermal equilibration. The cell has a U
shape, and to load the solution, one should use two 1 mL
pipettes whose tips have been modified to have a 1 cm soft
Teflon ending that whose radius perfectly fits to the DSC cell
sample entrance to allow good sealing. Even if the cell sample
volume is of only 300 μL, it is ideal to use a volume of at least
500 μL for the loading procedure as this will reduce the possi-
bility of introducing air bubbles that can be deleterious for the
measurements. After loading a cap is placed in one of the two
openings of the cell; the other opening is left open. A minimum
of two cycles of heating/cooling should be performed, more if
they differ (no stability reached). A flat line (no heat exchange)
should be observed. If this is not the case, this could arise from
several factors including the cell that is not properly cleaned,
the existence of buffer heat variations with temperature, or
others.
2. After blank measurement, the buffer loaded in the sample cell is
removed with a pipette, and the lipid sample is introduced in
the cell by the same method described above (no need to
change the solution in the reference cell). The same scan para-
meters used for the blank should be applied here. Typically, a
scan rate of 1 C/min is used at the temperature range to be
investigated with several (three to four) alternating heating and
cooling scans to allow the system to reach equilibrium (more
scans can be added if needed). Because the data in the first and
last 5 C of the scan range is often non-exploitable due to
baseline problems, one should keep that in mind and broaden
the temperature scan to take that into account. There is no
need to unload and load the reference cell since the buffer
solution used in this measurement is the same as in the blank.
Indeed the reference cell only needs to be filled once at the
beginning of a series of measurements. If several sets of experi-
ments are being run with the same buffer in the reference cell,
one needs to insure that no evaporation had occurred from that
cell. Even if each cell is capped in one of the two openings, the
other remains uncapped, so evaporation is possible after long
time periods.
3. Then to investigate the effect of the peptide on the lipid phase
transition, the sample containing both the lipid and the peptide
(at the appropriated P/L ratio, see Note 4) is loaded in the
sample cell, and the same parameters (temperature scan range
and number of heating and cooling scans) should be applied. It
is important to test whether the peptide alone undergoes ther-
mal reactions for the temperature range of the measurement.
This is usually not the case for small peptides (contrarily to large
proteins), as their unfolding does not involve high enough
thermal changes to be observed by DSC at the low concentra-

tions used for these experiments (below mM range).
4. After performing a complete set of experiments (blank, lipid
and lipid with peptide sample), the sample cell needs to be
cleaned. The cleaning solution is flowed through the cell by
connecting tubing that establishes a circuit between the cell
entrance and the washing solution through a vacuum system.
This ensures a continuous and fast flow of solution through the
cells. A solution of detergent in water, typically 200 mL of 1%
Hellmanex, is passed first followed by thorough washing with
1 L of Milli-Q water. This cleaning procedure is sufficient for
typical lipid and peptide samples. If this cleaning procedure is
found to be insufficient, harsher cleaning conditions such as
4 M sodium hydroxide followed by 50% formic acid can be
applied.
5. Ideally the cells should never be left empty to minimize hydra-
tion problems that occur when cells are filled after being left
dry for long periods of time. Therefore, one can load either
water or the reference buffer in the cell. The caps can be
cleaned with detergent solution and plenty of water. Be careful
about organic solvents as some cap materials are not compati-
ble with those resulting in changes in the shape and size of the
caps. For troubleshooting, see Note 5.
3.3 Data Analysis Data analysis for data obtained with a DSC TA Instrument is
and Interpretation: performed with the fitting program CPCALC provided by CSC.
Examples Other instruments usually provide their own software with
instrument.
1. The baseline (blank) should be subtracted from the sample
data. To avoid the introduction of different data treatment
from the user between different data sets, it is advisable to use
a flat baseline treatment and to treat the data within similar
temperature scans. It is best to start with the lipid alone for
which thermodynamic phase transition data has been well
reported in the literature, so that data can be compared. After
data subtraction with baseline, the following parameters are
obtained regarding the different lipid phase transitions (pre-
and main phase temperature transition for lamellar lipids):
phase transition temperature, enthalpy, and entropy associated
with the transition.
In the case where a mixture of lipids is used or a peptide is
added to the liposomes, the DSC signal can be broadened or
even splitted in a “two-peak” transition which could corre-
spond to a domain formation (lipid domain or peptide-poor/
peptide-rich domain). Data analysis software can be used to
perform the deconvolution of the thermogram and identify the

transition parameters of the different domains.
2. The exact same procedure is performed for the thermograms
corresponding to the lipid in presence of peptide.
3.4 Information From the parameters obtained after data analysis presented above,
Obtained from DSC information about the mode of interaction of the peptides with the
Analysis lipid membrane is obtained. Here a short summary is provided
about the different parameters and their meaning:
l Modification of the phase transition temperature. This indicates
that the peptide changes the lipid physicochemical properties
that are responsible for such transition. For example, if the
peptide decreases the Tm of a gel to fluid phase transition, it
means that the peptide favors the transition and thus has a
fluidizing effect on the membrane.
l Changes in the area of the transition peak. Since the area of the
transition peak is directly correlated with the enthalpy of the
transition, a decrease in the area indicates that the peptide per-
turbs the phase transition and decreases the enthalpy. For a gel to
fluid phase transition, this indicates that the peptide perturbs the
fatty acid chain packing and decreases the van der Waals interac-
tion due to intercalation between the fatty acid chains. In certain
cases the phase transition peak is completely abolished which is
very common for the pre-transition that is sensitive to molecular
interactions.
l Changes in the transition peak FWHM. This parameter is a
measure of the cooperativity of the transition; the sharper the
transition is, the more cooperative the transition. This parameter
is directly correlated with the changes in the peak area, and again
an increase of this parameter reflects peptide insertion in
between the fatty acid chains.
l Changes in the shape of the transition peak. Besides reflecting
changes in the cooperativity of the transition, this parameter is
related with homogeneity. The de-doubling of a single transition
peak and/or appearance of new transition peaks is indicative of
species heterogeneity. If the peptide does not distribute homo-
geneously among all lipids, the appearance of two-phase transi-
tion peaks corresponding to peptide-poor and peptide-rich
domains occurs. Usually the sharper transition peak is associated
with the peptide-poor lipid domains, and the broader transition
is attributed to peptide-rich lipid regions.
Alternatively, the appearance of new transition peaks can be due
to peptide-induced appearance of new lipid supramolecular
structures (liposomes of different size, micelles, etc.).
l Relation between main transition ΔH changes upon increasing
concentration of peptide. By plotting this data and extrapolating
the line, at ΔH ¼ 0 one can obtain the number of lipid molecules

removed from the cooperative chain-melting transition by each
molecule of peptide [8].
For more in-depth information, the reader is directed to exten-
sive review articles on the subject [8, 9, 21].
3.5 Unusual While most commonly single-lipid model systems are investigated
Applications/Cases by DSC, the method can be applied to the study of lipid mixtures.
3.5.1 Study of Lipid
Mixtures
3.5.2 Following Lipid In the context of binary lipid mixtures, DSC has been used to
Domain Formation follow domain formation in lipid membranes. It is well-known
and Peptide Partition into that cellular lipid membranes are not laterally homogeneous and
Different Domains that domains exist that either have morphological different struc-
tures or that only differ in their physicochemical properties such as
membrane ordering properties and fluidity, as it is the case for lipid-
ordered and lipid-disordered domains. DSC has been used to study
domain formation, mainly through the construction of phase dia-
grams and their interpretation in terms of phase miscibility. In view
of phase miscibility, DSC can be useful to follow lipid mixtures that
are miscible or not. In the first case, miscible lipid mixtures can
result in single-lipid phase transitions (that can become broader)
when lipids have Tm values that are close. For example, when
mixing lipids with different fatty acid chain lengths, lipids with
chain lengths that differ by less than four carbons are not miscible
[22, 23] and result in very well-separated phase transitions. Both
miscible and nonmiscible lipid mixtures can be interesting tools to
study the mechanism of action of membrane-active peptides: (1) In
the case of miscible lipid mixtures, DSC can be used to follow the
peptide effect in lipid reorganization, namely, the preferential
recruitment of one of the lipids in the mixture; (2) for nonmiscible
lipids that result in well-separated phase transitions observed by
DSC, the preferential interaction of a peptide with one of the two
lipids can be determined by the changes induced in each transition
after peptide addition. Moreover, some peptides have the capacity
to improve lipid miscibility (for details, see refs. 24–27).
4 Notes
1. Ideal buffers for DSC measurements should undergo minimal

heat capacity variation with temperature. Most commonly used
buffers in the laboratory are acceptable as Tris, glycine, phos-
phate, etc. Buffers whose pKa varies considerably with temper-
ature such as HEPES can be problematic. One point that
absolutely needs to be considered is that the buffer composi-

tion must be absolutely the same in the reference and in the cell
as very small variations in ion content can greatly affect the heat
capacity and data measurements.
2. The most commonly used lipid model systems for DSC mea-
surements are MLVs, because it is a simple system that can
easily be prepared in all laboratories without the need for
special equipment. Additionally MLVs are very stable in time,
and the phase transitions produced are very sharp and due to
the high cooperativity of the transition. Another possibility is
to use large unilamellar liposomes (LUVs). These systems are
less stable than MLVs but still stable long enough for the time
required for the measurements to be acquired. One advantage
in using LUVs is related with the fact that the P/L ratio can be
properly controlled because in LUVs the ratio of lipid present
in the outer versus inner leaflet is known. For MLVs, since their
size and amount of bilayers per object are heterogeneous, the
P/L ratio cannot be properly determined. The only exception
to that is if the peptides investigated are able to cross all lipid
layers and distribute homogeneously between the different
membranes. Alternatively, it is also common to add peptides
to lipids before formation of liposomes, the peptide is added at
the moment of preparing the lipid film, and therefore the
peptide becomes evenly distributed among the different lipid
layers. This can be advantageous if the peptide is not easily
dissolved in polar solvents. In our laboratory we have privileged
the addition of peptide to lipid after liposome formation
because this procedure resembles best the biological system.
3. Regarding the composition of the lipid model system, one
needs to choose a lipid whose phase transition temperature is
above 0 C (the instrument cannot record below that unless a
special solvent is used that does not freeze at this temperature).
For lipid model systems composed of a single type of lipid and
for the investigation of gel/fluid phase transition, 1 mg of lipid
is enough for a set of experiments. If lipid mixtures are used,
the amount of lipid needed for DSC measurements may have to
be adapted depending on the lipid phase transition and
whether the lipids are miscible or not. Keep in mind that in
DSC one cannot use very complex lipid mixtures because this
will result in very broad and shallow phase transitions. For
lipids that are totally nonmiscible, in principle their phase
transition properties are kept, and thus there is no need to
increase the amount of lipid used. If lipids are miscible, the
resulting phase transition is going to become broader and thus
less well resolved; therefore, lipid concentrations must be
increased so that the transition can be properly followed. Lipids
such as cholesterol when added to phospholipids have the
tendency to greatly broaden the transition, so again higher lipid

concentrations are necessary. Similarly, if one is to investigate
other lipid phase transitions than the gel/fluid, for example,
the transition of lamellar (Lα) to hexagonal (HII) phase transi-
tion, because smaller energy changes are implicated in such
phase transitions (smaller transition peak), then the amount
of lipid must be increased. In the case of the Lα to HII phase
transition of DiPoPe (dipalmitoleoyl phosphatidylethanol-
amine), 10 mg of the lipid was used in reported studies from
our laboratory for a reasonably quantifiable transition to be
monitored [28].
4. DSC studies on P/L interactions have used P/L ratios varying
from 1/500 to 1/10. The P/L ratio at which an effect will be
measurable by DSC depends on the nature and extent of the
peptide-induced lipid perturbation which is related with the
class of membrane-active peptide used. In general when the
appropriated lipid is chosen, considerable peptide effects can be
observed at P/L ratios of 1/50. Care must be taken with the
use of very high peptide concentrations because precipitation
may occur perturbing DSC signal.
5. This is not an exhaustive list but rather a summary of most
common problems that can be encountered during measure-
ments and how to solve them. Some of them have been briefly
mentioned along the protocol.
(a) Buffer/buffer scans are inconsistent. Heat capacity
increases in subsequent scans. This can be due to the
presence of air bubbles in the solution. The solution
should be degassed and reloaded again.
(b) There is an effective Y-offset in the heat capacity data. This
can be caused by pressure variation during data acquisi-
tion. This can result from opening/reclosing the cell to
introduce the sample. The pressure is never exactly the
same after replacing the pressure cap. This can also result
from caps that are getting overused and that do not tightly
close anymore.
(c) Sample scans are not similar between them (when com-
paring among heating or among cooling). This can just be
due to the fact that the sample is not yet equilibrated and
that temperature scans affect sample properties and
homogeneity. The solution is to continue heating/cool-
ing scans until the thermograms are comparable, meaning
that the solution reached thermal equilibrium.
(d) The pressure does not stabilize. This can be due to several
problems: buffer volume in the cells is too low, and the
cap used to close of the apertures is old and does not cap
correctly.
References
1. Cullis PR, Fenske DB, Hope MJ (1996) Physi- 14. Garidel P, Blume A (2000) Miscibility of
cal properties and functional roles of lipids in phosphatidylethanolamine-
membranes. In: Vance DE, Vance JE (eds) phosphatidylglycerol mixtures as a function of
Biochemistry of lipids, lipoproteins and mem- pH and acyl chain length. Eur Biophys J
branes. Elsevier, Amsterdam, pp 1–32 28:629–638
2. Israelachvili JN, Mitchell DJ, Ninham BW 15. Raudino A (1995) Lateral inhomogeneous
(1977) Theory of self-assembly of lipid bilayers lipid membranes: theoretical aspects. Adv Col-
and vesicles. Biochim Biophys Acta loid Interf Sci 57:229–285
470:185–201 16. Almeida PF (2009) Thermodynamics of lipid
3. Lee AG (1977) Lipid phase transitions and interactions in complex bilayers. Biochim Bio-
phase diagrams. I. Lipid phase transitions. Bio- phys Acta 1788:72–85
chim Biophys Acta 472:237–281 17. Riske KA, Barroso RP, Vequi-Suplicy CC,
4. Lee AG (1977) Lipid phase transitions and Germano R, Henriques VB et al (2009) Lipid
phase diagrams. II. Mictures involving lipids. bilayer pre-transition as the beginning of the
Biochim Biophys Acta 472:285–344 melting process. Biochim Biophys Acta
5. McElhaney RN (1982) The use of differential 1788:954–963
scanning calorimetry and differential thermal 18. Lichtenberg D, Freire E, Schmidt CF,
analysis in studies of model and biological Barenholz Y, Felgner PL et al (1981) Effect
membranes. Chem Phys Lipids 30:229–259 of surface curvature on stability, thermody-
6. McIntosh TJ (1996) Hydration properties of namic behavior, and osmotic activity of dipal-
lamellar and non-lamellar phases of phosphati- mitoylphosphatidylcholine single lamellar
dylcholine and phosphatidylethanolamine. vesicles. Biochemistry 20:3462–3467
Chem Phys Lipids 81:117–131 19. Mason JT, Huang C, Biltonen RL (1983)
7. Epand RM, Bryszewska M (1988) Modulation Effect of liposomal size on the calorimetric
of the bilayer to hexagonal phase transition and behavior of mixed-chain phosphatidylcholine
solvation of phosphatidylethanolamines in bilayer dispersions. Biochemistry
aqueous salt solutions. Biochemistry 22:2013–2018
27:8776–8779 20. Rouser G, Fkeischer S, Yamamoto A (1970)
8. McElhaney RN (1986) Differential scanning Two dimensional then layer chromatographic
calorimetric studies of lipid-protein interac- separation of polar lipids and determination of
tions in model membrane systems. Biochim phospholipids by phosphorus analysis of spots.
Biophys Acta 864:361–421 Lipids 5:494–496
9. Seelig J (2004) Thermodynamics of lipid- 21. Lohner K, Prenner EJ (1999) Differential scan-
peptide interactions. Biochim Biophys Acta ning calorimetry and X-ray diffraction studies
1666:40–50 of the specificity of the interaction of antimi-
10. Heerklotz H (2004) The microcalorimetry of crobial peptides with membrane-mimetic sys-
lipid membranes. J Phys Condens Matter tems. Biochim Biophys Acta 1462:141–156
16:441–467 22. Leidy C, Wolkers WF, Jorgensen K, Mouritsen
11. Jimenez-Monreal AM, Villalain J, Aranda FJ, OG, Crowe JH (2001) Lateral organization
Gomez-Fernandez JC (1998) The phase and domain formation in a two-component
behavior of aqueous dispersions of unsaturated lipid membrane system. Biophys J
mixtures of diacylglycerols and phospholipids. 80:1819–1828
Biochim Biophys Acta 1373:209–219 23. Shimshick EJ, Kleemann W, Hubbell WL,
12. Epand RM, Bach D, Epand RF, Borochov N, McConnell HM (1973) Lateral phase separa-
Wachtel E (2001) A new high-temperature tions in membranes. J Supramol Struct
transition of crystalline cholesterol in mixtures 1:285–294
with phosphatidylserine. Biophys J 24. Joanne P, Galanth C, Goasdoue N, Nicolas P,
81:1511–1520 Sagan S et al (2009) Lipid reorganization
13. Lewis RN, Zhang YP, McElhaney RN (2005) induced by membrane-active peptides probed
Calorimetric and spectroscopic studies of the using differential scanning calorimetry. Bio-
phase behavior and organization of lipid bilayer chim Biophys Acta 1788:1772–1781
model membranes composed of binary mix- 25. Epand RM (2007) Detecting the presence of
tures of dimyristoylphosphatidylcholine and membrane domains using DSC. Biophys Chem
dimyristoylphosphatidylglycerol. Biochim Bio- 126:197–200
phys Acta 1668:203–214
26. Epand RF, Wang G, Berno B, Epand RM the lateral domain organization of lipid
(2009) Lipid segregation explains selective tox- bilayers. Biochim Biophys Acta 1328:125–139
icity of a series of fragments derived from the 28. Alves ID, Goasdoue N, Correia I, Aubry S,
human cathelicidin LL-37. Antimicrob Agents Galanth C et al (2008) Membrane interaction
Chemother 53:3705–3714 and perturbation mechanisms induced by two
27. Polozov IV, Polozova AI, Molotkovsky JG, cationic cell penetrating peptides with distinct
Epand RM (1997) Amphipathic peptide affects charge distribution. Biochim Biophys Acta
1780:948–959
Chapter 2
Protocols of IATC, DSC, and PPC: The Multistate Structural

Transition of Cytochrome c
Shigeyoshi Nakamura and Shun-ichi Kidokoro
Abstract
The recent development of high-precision calorimeters allows us to monitor the structural transition of
biomolecules by calorimetry and thereby characterize the thermodynamic property changes accompanying
three-dimensional structure changes. We developed isothermal acid-titration calorimetry (IATC) to evalu-
ate the pH dependence of protein enthalpy. Using the double deconvolution method with precise differ-
ential scanning calorimetry (DSC), we revealed that the MG state is an equilibrium intermediate state of the
reversible thermal three-state transition of the protein, and we successfully determined its volumetric
properties by pressure perturbation calorimetry (PPC). Our findings underscore the importance of a precise
calorimetry and analysis model for protein research.
Key words Differential scanning calorimetry, Pressure perturbation calorimetry, Cytochrome c, Iso-
thermal acid-titration calorimetry
1 Introduction
Calorimetry is the strongest method for evaluating the thermody-

namic parameters in the structural transition of proteins. Over the
last several decades, calorimetry has improved our thermodynamic
knowledge of protein stability by providing a direct method to
measure thermodynamic functions [1–15]. In particular, precise
differential scanning calorimetry (DSC) has enabled us to deter-
mine the enthalpy of a protein molecule as a function of tempera-
ture. The Gibbs free energy, in turn, can be evaluated from the
enthalpy. The heat capacity change is understood to be mainly the
result of a change in the hydration of the protein molecule accom-
panying the structural transition and is important for understand-
ing the mechanism of protein stability [16–18].
Volumetric parameters such as the partial volume and the ther-
mal expansion coefficient are sensitively affected by the hydration
state of protein surfaces. Pressure perturbation calorimetry (PPC) is
Eric Ennifar (ed.), Microcalorimetry of Biological Molecules: Methods and Protocols, Methods in Molecular Biology, vol. 1964,
https://doi.org/10.1007/978-1-4939-9179-2_2, © Springer Science+Business Media, LLC, part of Springer Nature 2019
17
18 Shigeyoshi Nakamura and Shun-ichi Kidokoro
an effective technique for evaluating the thermal expansion coeffi-

cient, αp, of biomaterials [19–22].
Isothermal titration calorimetry (ITC) is also a powerful
method for measuring the dependence of the protein stability on
the solvent composition under a constant temperature [3, 23,
24]. Isothermal acid-titration calorimetry (IATC) was developed
to evaluate the thermodynamic parameters in the pH-induced
structural transition [25, 26].
In this chapter, we introduce the protocols of IATC, DSC, and
PPC in the thermal three-state transition of cytochrome c [27, 28].
2 Materials
2.1 Preparation In this example, an ITC unit of MCS system (MicroCal, North-
for Isothermal Acid- ampton, USA) is used.
Titration Calorimetry 1. Blank solution: 20 mM KCl. Prepare 2 L of blank solution.
(IATC) Add 3.0 g KCl to 1.8 L water (pure Milli-Q water). Adjust the
pH to about 7 by adding NaOH solution (see Note 1). Make
up to 2 L of blank solution with water. Store at 4 C.
2. HCl solution for acid titration: 20 and 400 mM HCl solutions
in 20 mM KCl. Dilute 1 M HCl solution with water to make a
20 mM and a 400 mM HCl solution (see Note 2).
3. Protein solution: Dissolve lyophilized powder of cytochrome
c as 0.5 mg/mL solution with blank solution. Prepare about
30 mL of protein solution for one set of isothermal titration
calorimetry (ITC) measurements (two ITC measurements with
20 mM and 400 mM HCl) and the pH measurements (two pH
measurements with 20 mM and 400 mM HCl) at one temper-
ature. Protein solutions of 3 mL and 10 mL are needed for one
set of ITC and pH measurement, respectively. Store at 4 C.
4. Dialysis: Dialyze the above-described protein solution with a
Spectra/Por dialysis membrane (132,660; cutoff molecular
weight, 6000–8000; Spectrum Laboratories, Rancho Domin-
guez, CA) at 4 C for 1–2 days against a total 2 L of blank
solution. Dialyze 15 mL of protein solution four times against
500 mL of blank solution. The times of dialysis are over 4 h
(first dialysis) and over 8 h (2nd–4th dialysis). After each dialy-
sis, replace 500 mL blank solution with fresh solution for the
next dialysis. Also after final dialysis, collect a protein solution
and store it at 4 C.
5. Subject the protein solution to ultrafiltration with a MolCut
ultra filter unit (USY-20; Advantec, Tokyo, Japan) just before
the IATC measurement (see Note 3).
Calorimetry of the Cytochrome c 19
2.2 Preparation 1. Blank solution: 50 mM acetate buffer at pH 4.1. Prepare 2 L of

for DSC, PPC, buffer solution. Add 6.0 mL acetic acid to 1.8 L of water (pure
and Density Milli-Q water). Adjust the pH with NaOH to 4.1. Make up to
Measurement 2 L of blank solution with water. Store at 4 C.
2. Protein solution: Dissolve the lyophilized powder of proteins as
a 20 mg/mL solution with blank solution (50 mM acetate
buffer at pH 4.1). Prepare about 6 mL of protein solution.
Store at 4 C.
3. Dialysis: Dialyze the protein solution with a Spectra/Por dialy-
sis membrane (132,660; cutoff molecular weight, 6000–8000;
Spectrum Laboratories) at 4 C for 1–2 days against a total 2 L
of blank solution. Dialyze 6 mL protein solution four times
against 500 mL blank solution. The times of dialysis are over
4 h (first dialysis) and over 8 h (2nd–4th dialysis). After each
dialysis, replace 500 mL blank solution with fresh solution for
the next dialysis. Also after final dialysis, collect a protein solu-
tion and store it at 4 C.
4. Subject the protein solution to ultrafiltration with a MolCut
ultra filter unit (USY-20; Advantec) just before the DSC and
PPC measurements (see Note 3).
5. Determine the concentration of protein solution with a spectro-
photometer by using an extinction coefficient of
ε409 ¼ 9.197 104/M/cm. The concentration of cytochrome
will become about 18 mg/mL after filtering. Use 18 mg/mL
cytochrome c for the PPC and density measurement (see Note 4).
Dilute the cytochrome c solution by adding buffer to adjust to
about 1 mg/mL of cytochrome c for DSC.
6. Perform the complete degassing of the protein solution by
several minutes of aspiration using a membrane pump, and
simultaneously sonicate the solution using a small sonication
device just before the measurements of DSC, PPC, and density.
7. Perform the differential scanning calorimetry (DSC) experi-
ments of cytochrome c with a highly sensitive differential scan-
ning calorimeter, MicroCal VP-DSC (Malvern Instruments
Ltd., Worcestershire, UK).
8. DMA5000 high-precision vibrating tube densitometer (Anton
Paar, Graz, Austria).
9. Perform the pressure perturbation calorimetry (PPC) experi-
ments of cytochrome c with a highly sensitive differential scan-
ning calorimeter, MicroCal VP-DSC, equipped with a PPC
accessory pressurizing system (Malvern Instruments Ltd.).
20 Shigeyoshi Nakamura and Shun-ichi Kidokoro
3 Methods
3.1 IATC IATC is the calorimetric method used to evaluate the thermody-
namic parameters in the acid-induced structural transition of pro-
tein. IATC measurement consists of ITC and pH measurements.
During the IATC measurement, acid titrations of
low-concentration HCl (20 mM HCl) and high-concentration
HCl (400 mM HCl) to the cytochrome c solution are performed
to evaluate the thermodynamic parameters precisely (see Note 2).
3.1.1 ITC for IATC 1. Determine the concentration of the protein solution with a
spectrophotometer by using an extinction coefficient of
ε409 ¼ 9.197 104/M/cm. Use 0.5 mg/mL cytochrome
c in 20 mM KCl as the sample solution (see Subheading 2.1,
item 2).
2. Perform the complete degassing of the protein solution for
several minutes by aspiration with a membrane pump while
simultaneously sonicating the solution with a small sonication
device (see Note 5).
3. ITC measurement for the protein solution with 20 mM HCl:
Perform the 20 mM HCl titration to protein solution using an
isothermal titration calorimetry. First, load the protein solution
in the ITC sample cell. The cell volume is 1.368 mL. Perform
the titration with injections of 2 μL (1st–20th injections), 5 μL
(21th–35th injections), and 10 μL (35th–50th injections) in
each of 20 mM HCl solution in 20 mM KCl using a 250 μL
syringe (see Note 6). Before each experiment, wash the ITC cell
several times with a blank solution. In the present experiments,
the ITC measurements were performed at 40 C.
4. ITC measurement for the blank solution with 20 mM HCl:
Perform the control experiment (20 mM HCl titration to the
blank solution) in the absence of protein solution throughout
the same pH range by using ITC.
5. ITC measurement for protein solution with 400 mM HCl:
Perform the 400 mM HCl titration to protein solution using
a microcalorimeter. Conduct the titration using injections of
2 μL (1st–20th injections), 5 μL (21th–35th injections), and
10 μL (35th–50th injections) in each of 400 mM HCl solution
in 20 mM KCl using a 250 μL syringe. Before each experiment,
wash the ITC cell several times with a blank solution.
6. ITC measurement for the blank solution with 400 mM HCl:
blank solution) in the absence of protein solution throughout
the same pH range of the sample measurement by using ITC.
Calorimetry of the Cytochrome c 21
3.1.2 pH Measurement 1. Calibration of pH meter: Perform the three-point calibration

for IATC of the pH meter using standard pH solutions of pH 2, pH 4,
and pH 7. Correct the observed pH values of the protein
solution with the second-order polynomial function, the coef-
ficients of which are determined by using the pH values of three
standard solutions at the experimental temperature.
2. pH measurement for the protein solution with 20 mM HCl:
Measure the pH of 20 mM HCl titration with the protein
solution by using a glass electrode and a pH meter. For the
pH measurements, use a solution identical to that used for the
ITC measurements. Use a 10 mL initial volume of the protein
solution and then determine the injection volumes as the ratios
between the initial and the injection volumes became identical
to those of each corresponding injection in the ITC measure-
ments. In the case described in Subheading 3.1.1, step 3, the
ITC conditions were as follows: ITC cell volume, 1.368 mL;
injection volumes, 2 μL (1st–20th injections), 5 μL (21th–35th
injections), and 10 μL; and injection volumes of the pH mea-
surements, 14.6 μL (1st–20th injections), 36.5 μL (21th–35th
injections), and 73 μL (35th–50th injections). Perform these
injections manually by using a pipetman (see Note 7). Keep the
temperature of the titration vessel constant in a handmade glass
bath with circulating water from a thermostat water bath.
Record the pH values after each titration.
3. pH measurement for the blank solution with 20 mM HCl:
blank solution) in the absence of a protein solution throughout
the same pH range by using the ITC measurements of the
blank experiment. Use the same initial volume and injection
volume as under the experimental conditions for the pH mea-
surements of the protein solution. Record the pH values after
each titration.
4. pH measurement for protein solution with 400 mM HCl:
Perform the pH measurement of 400 mM HCl titration with
the protein solution by using a glass electrode and a pH meter
in the same manner as for the pH measurement of the sample
solution with 20 mM HCl (see Subheading 3.1.2, step 2). Use
the same solution for the pH measurements as used for the ITC
measurements. For the pH measurements, the initial volume of
the protein solution is 10 mL, and the injection volumes are
determined as the ratios between the initial and the injection
volumes become identical to those of each corresponding
injection in the ITC measurements. In the case of the experi-
mental conditions for ITC (see Subheading 3.1.1, step 5), the
Another random document with
no related content on Scribd:
the general cultivation of the country, its roads, carriages, and modes
of travelling; its canals, ships, trade, wars, &c.; the bravery of the
men, and the beauty of the women, with the richness of their dress;
and that this prosperous state of things resulted from its good
government, the king encouraging people from all parts of the world
to come to England and trade, and sending his own people to visit
the most distant corners of the earth to see what in every country
might be of use in England. It was midnight before we parted, and
then I had to send them away, telling them we had to rise early in the
morning. Burgho is only a day’s journey from this, and the natives of
that country often come and steal people from the neighbouring
towns to sell into slavery. At 7 A.M. left Laydoo; the country but little
cultivated, thin woods, soil a red clay mixed with lumps of iron-stone,
none being larger than three feet on each side. At 8.30 halted at the
village of Leogalla, inhabited by Fellatahs, who kindly brought us
sweet milk to drink. At 10 halted at the village of Bongbong, where
the carriers got their breakfast of eko, or accassan, which is made of
millet meal, first steeped in water until sour, then boiled like a thick
paste, and then mixed with warm or cold water for a drink, or eaten
without water for food: it is very wholesome. The village of Bongbong
is walled. At 10.30 started—passed a burnt village—the road winding
—country woody, forming gentle dale and down; a strong harmattan,
or north wind, blowing. At noon halted at Atepa; got quarters in the
caboceer’s house, where we were supplied with yams, fowls, a goat,
turkey, &c., also a large pig, which we gave to Abaco, the king’s
messenger. The caboceer was very inquisitive about England.
Saturday, 21st.—Morning cold and clear. This night the
thermometer has been as low as 55° in the open air. The coldest
time in this country, as, I believe, in all others, is the hour before day-
light, or rather before sunrise. The town is large and populous,
containing certainly above six thousand; it is surrounded by a belt of
trees, rendered impenetrable by the crossing thorny creepers,
through which there is only a narrow pass at the gates. The country,
for a couple of miles outside the gate, well cultivated. At 9.15 entered
the walled town of Namah, the road now winding and woody;
changed carriers; and at 9.30 left Namah. The country plain, and a
clay soil. At 10 A.M. crossed a stream called Juffee or Moussa which
runs into the Quorra at, or opposite to, Nyffee. At 11.30 arrived at the
walled town of Leobadda: there is a range of broken rocks, like an
immense wall, on the east side of which the town is built. We were
accompanied hither by the caboceers of Atepa and Namah, with all
their train, to guard us from the Burgho robbers who frequent the
road, as the king of that country has his capital only one day’s
journey with a horse from this place. Passed two ruined villages; the
road woody and winding. We gave the two caboceers a dram before
starting, as they had been very kind to us.
Sunday, 22d.—Clear and cold; north wind during the night;
thermometer, 56°. The town of Leobadda is situated on the east side
of a ridge of granite, the tops of which are broken into large masses,
some of them forming the most grotesque figures imaginable; they
run in a direction from north-east to south-west, and are from fifty to
sixty feet above the plain, and join the hills to the south and east.
Leobadda contains about one hundred and fifty houses, with from
thirty to forty souls in each; it is walled; the inhabitants are poor, but
civil; we were well supplied with provisions, as before, and in
addition had milk. The caboceer told us that Kiama, the capital of
Borgho, was only one day’s journey by a horse from his town; that
these people were only a band of thieves; that their country was
small, but independent; that they infested the roads of Youriba, and
stole all they could catch.
At 7 A.M. started, accompanied by the caboceer and a great
number of attendants. The country well cultivated for a little way
outside the town. We met upwards of six hundred men, women, and
children, carrying loads; they had travelled all night, and were
guarded by men with bows and arrows and swords, ten or twelve
armed men marching between each fifty; the road woody, but the
trees low and stunted. Here, for the first time, I saw the small stunted
accacia. The soil red clay. Passed several villages that had been
destroyed by the Fellatahs, some very large. The shady trees are
now desolate, the walls covered with weeds. After closing with the
range of rocks we entered a beautiful valley in the midst of them,
planted with large shady trees and bananas, having green plots, and
sheets of water running through the centre, where the dingy beauties
of Tshow were washing their well-formed limbs, while the sheep and
goats were grazing around on the verdant banks. After passing this
lovely valley we crossed another ridge of rocks, and at 9.15 arrived
at the town of Tshow, where, after getting housed, we turned to and
cleaned our arms, as they say the road is infested with robbers. We
afterwards heard that the king of Eyeo was going to send an escort,
and was quite rejoiced at our near approach. Got a specimen of the
Tsheu fruit and leaves: the fruit is the size of a large pear, having a
stone inside, covered with a pulpy cream-coloured substance, which
is good to eat. The stone is said to be poisonous; the outer rind of
the fruit is put into their soup.
After sunset a caboceer arrived from the king of Katunga or Eyeo.
His attendants, horse and foot, were so numerous that every corner
was filled with them, and they kept drumming, blowing, dancing, and
singing all night. The caboceer waited on us, and after shaking
hands with us, rubbed all his face and body over, that his hands, by
touching ours, might impart our blessing to his face, head, and body.
They were well dressed, and said the king of Eyeo was most anxious
to see us: they had a great deal of natural good-breeding; and on my
saying I had brought only a small present, they replied, if I had
brought nothing the king of Eyeo would be glad to see us. Sent them
a flask of rum. They kept firing all night.
Monday, 23d.—The town of Tshow was all bustle and hubbub with
the great men and their attendants, the large grooms and their little
horses. I left it early, being a poor place, with a good wall, and may
contain four thousand inhabitants. Perhaps I might call them poor, as
they had not fed us so well as we had been wont to be fed; but
considering the ravenous host which came to escort us to Katunga,
and who five pretty freely wherever they can, this deficiency on our
side is easily accounted for. The road through which we passed was
wide, though woody, and covered by men on horseback, and
bowmen on foot. The horsemen armed with two or three long spears
hurrying on as fast as they could get us to go; horns and country
drums beating and blowing before and behind; some of the
horsemen dressed in the most grotesque manner; others covered all
over with charms. The bowmen also had their natty little hats and
feathers, with the jebus, or leathern pouch, hanging by their side.
These men always appeared to me to be the best troops in this
country and Soudan, from their lightness and activity. The horsemen
however are but ill mounted; the animals are small and badly
dressed, their saddles so ill secured, and the rider sits so clumsily on
his seat, that any Englishmen, who ever rode a horse with an
English saddle, would upset one of them the first charge with a long
stick.
We were also accompanied by great numbers of merchants or
traders. At 9 A.M. halted to the southward of a village called
Achoran, until the baggage should come up; but the heavy packages
not coming soon, the caboceers thinking we would be too much
exhausted before we got into Katunga, sent us off with a proper
escort, waiting themselves for the baggage. At 9.30 left the shade,
and went on as hard as the horses could possibly walk; the road
winding and woody; clay iron-stone, resembling lava, in the spaces
between the granite, great blocks of which appeared on each side of
the ravines and low hills. We had from the top of one of the ridges
some beautiful views to the east, of fine wooded valleys and low
rugged bare hills in the back ground. At 10.20 crossed a stream
running to the Quorra, which is only a journey of three days distant.
Here we drank and gave our horses water; passed the ruins of two
towns burned by the Fellatahs. At 11.30, from the top of a high ridge,
on which were the ruins of a clay wall, we saw the city of Katunga or
Eyeo. Between us and it lay a finely cultivated valley, extending as
far as the eye could reach to the westward; our view intercepted to
the eastward by a high rock, broken into larger blocks, with a
singular top; the city lying, as it were, below us, surrounded and
studded with green shady trees, forming a belt round the base of a
rocky mountain, composed of granite, of about three miles in length,
forming as beautiful a view as I ever saw.
At 12.15 we entered the north gate of Katunga; there was a small
Fetish house outside the gate, and a few others inside. We halted,
and went into one of the caboceer’s houses until the baggage and
escort came up. Here we got accassan; and Abaco’s wife was
cooking a little country soup for us, but the pot broke on the fire just
as it was ready, and the house was in an uproar in an instant; only
for my interference they would have come to blows. Abaco was
ready to cry that he had disappointed us.
At 2 P.M. the baggage having all arrived, a message came from
the king to say that he wanted to see us. A band of music
accompanied us and the escort, with an immense multitude of men,
women, and children. As there was much open and cultivated
ground, the dust they caused almost suffocated us, though the
escort tried all gentle means to keep them off. At last after riding one
hour, which was full five miles, we came to the place where the king
was sitting under the verandah of his house, marked by two red and
blue cloth umbrellas, supported by large poles held by slaves, with
the staff resting on the ground. After the head caboceers had held
some conversation with the king, they came back to us, and I
thought they were talking about our prostrations. I told them if any
such thing was proposed, I should instantly go back; that all the
ceremony I would submit to should be to take off my hat, make a
bow, and shake hands with his majesty, if he pleased. They went
and informed the king, and came back and said I should make only
the ceremony I had proposed. We accordingly went forwards: the
king’s people had a great deal to do to make way amongst the
crowd, and allow us to go in regular order. Sticks and whips were
used, though generally in a good-natured manner; and I cannot help
remarking on this, as on all other occasions of this kind, that the
Youribas appear to be a mild and kind people, kind to their wives and
children and to one another, and that the government, though
absolute, is conducted with the greatest mildness. After we got as far
as the two umbrellas in front, the space was all clear before the king,
and for about twenty yards on each side. We walked up to the
verandah with our hats on, until we came into the shade, when we
took off our hats, made a bow, and shook hands; he lifting our hands
up three times, repeating “Ako, Ako,” (how do you do?) the women
behind him standing up and cheering us, calling out “Oh, oh, oh!”
(hurrah!) the men on the outside joining. It was impossible to count
the number of his ladies, they were so densely packed and so very
numerous. If I might judge by their smiles, they appeared as glad to
see us as their master. The king was dressed in a white tobe, or
large shirt, with a blue one under; round his neck some three strings
of large blue cut-glass beads; and on his head the imitation of an
European crown of blue cotton covered over pasteboard, made
apparently by some European, and sent up to him from the coast.
We waited about half an hour until all inquiries had been made
respecting our health and the fatigues of our journey. We were then
conducted by his chief eunuch and confidents to apartments in the
king’s house, and asked if we liked them. They certainly were very
good; but our servants would have been too far removed from us.
We looked out for one in which we could be more comfortably
stowed, and one by which both servants and baggage would be
under our own eyes; which is an important matter, in this country
especially, and never to be neglected, however good the servants
may be. After this we had dinner and tea, to which we had good new
milk; and when it got fairly dark we had a visit from the king in
person: he was attended by his favourite eunuch, the ladies
remaining outside. He was very plainly dressed, so that he would not
have been known outside but as one of the people, with a long staff
in his hand. He said he could not sleep until he saw us, but that we
should only talk about our health, and not about business now. After
a short stay he went away. We requested before he went that we
should be left undisturbed for two days, that we might rest from the
fatigues of our journey.
CHAPTER II.
RESIDENCE AT EYEO, OR KATUNGA, THE CAPITAL OF YOURIBA.
Tuesday, January 24th.—Early this morning the king paid us a

visit, accompanied by his favourite eunuch and Abaco the
messenger. He had received previous information that he wished to
have the presents intended for him this night; and such is the
crooked policy of these petty sovereigns of Africa, that no present
can be given, no business or transaction of importance can be done
openly: all must be conducted under the cover of night, and with the
greatest secrecy, from the highest to the lowest. We first began
inquiring after his health. I then told him that I was the king of
England’s servant, sent by his majesty to beg his acceptance of a
present, which then lay before me; that we had heard his (the king of
Youriba’s) name mentioned in England as a great king; that we now
experienced the truth of the report; that three white men, two of them
my companions, and one a servant, had died on the road; that
another of my companions was at Dahomey, to ask the king of that
country to allow him a passage through his dominions. I told him that
all the Youriba people had behaved well to us; that the caboceers of
the different towns through which we had passed supplied us with
every thing we wanted, especially the chief of Jannah, his friend,
who had shown the greatest attention to us, and had given us a
good man for a messenger, who had conducted us with safety and
attention to his majesty’s capital. Upon this the messenger was
ordered to make his prostrations, and his majesty rubbed his
shoulders with his hand. I then told him that the king of England
would be glad to make him his friend, and that whatever he, the king
of Youriba, might have occasion for would be sent from England by
one of the king’s ships to Badagry.
The king then replied in assuring us that we were truly welcome to
his country; that he had frequently heard of white men; but that
neither himself, nor his father, nor any of his ancestors had ever
seen one. He was glad that white men had come at this time; and
now he trusted his country would be put right, his enemies brought to
submission, and he would be enabled to build up his father’s house,
which war had destroyed. This he spoke in such a feeling and
energetic manner, and repeated it so many times, that I could not
help sympathising with him. He then said we were welcome to his
country, and he was glad to see us, and would have been so, even if
we had not a cowrie, instead of coming with our hands full, as we
had done; that he wanted nothing from white men, but something to
assist him against his enemies, and those of his people who had
rebelled against him, so as to enable him to reduce them to
obedience; that his slaves from Housa had joined the Fellatahs, put
to death the old, and sold the young; that he was glad to hear that all
his people had behaved well to us; that had any of them refused us
assistance, he should have sent an order to cut off their heads; that
the caboceer of Jannah was his slave, whom he put there to look
after that part of his dominions; that Badagry, Alladah, and Dahomey
all belonged to him, and paid custom for every ship that anchored
there: and he concluded by assuring us he wanted nothing but
assistance against his enemies; feelingly deploring the civil war
occasioned by his father’s death, the state of his country, and of his
capital, Katunga. He then asked us if we did not see the ruined
towns as we came along the road. “All these,” says he, “were
destroyed and burned by my rebellious Housa slaves, and their
friends, the Fellatahs.”
We now began to unfold and to deliver the presents. With the
umbrellas and gold-mounted cane he was much pleased; but for the
red and blue cloth, which, by some mistake, was common cloth for
soldiers’ coats, we had to make an apology. With all the others he
was highly pleased. Indeed, during our stay at Katunga he was
never seen without the cane.
After the delivery of the presents, I told him that the king, my
master, had sent me before on a mission to Bornou, in which country
and Housa I had passed two years; that the sultans and people of
these countries had behaved to me with the greatest kindness; and
that, having then understood that the path we were now going was
the nearest way to Bornou, the king of England had ordered me, as I
proceeded, to visit the king of Youriba, and to assure him of his
friendship, and to request him to give me a safe conduct to Nyffee,
from whence I might proceed to Bornou.
He seemed to hesitate much at this request, and consulted with
his minister what answer to give. After which, he said, that Nyffee or
Toppa was involved in civil war, caused by the death of the king, who
had left two sons, both of whom claimed the kingdom; that one son
had more of his countrymen on his side, but the other had called in
the assistance of the Fellatahs or Fellans, which made him doubt as
to my safety, in the event of his putting me into their hands. I told him
I was a servant of the king of England, and must go where he chose
to order me, and that, live or die, I must proceed; that I had nothing
to do with either party or with their wars; that all I wanted was a
passage over the Quorra into Nyffee, and hoped he would not refuse
me. After some further consultation with his counsellors, he said he
would despatch a messenger to open the road for me, and that he
would send me safely over the river.
Wednesday, 25th.—Early this morning the king sent me a present
of a large fat cow, a sheep, yams, &c. He had before sent us a goat,
yams, honey, and milk, night and morning.
The atmosphere here is so dry, that most of the instruments are
breaking and splitting. My only hygrometer was broken at Badagry.
The late Dr. Morrison’s barometers were fitted with ivory screws at
the bottom of the tube; they are all split, and rendered useless by the
heat: the plain tubes are the best; those with ivory or wooden scales
contract, and break the glass. The microscope is all in pieces, as
also several other instruments.
In the evening we had a visit from the king, to thank me for the
presents I had given him, and again to assure me of being welcome;
said that he wanted nothing, unless it was something that would
speedily cause the submission of the rebels. He said that he had
sent to his friend the king of Benin for troops to assist him in the war.
He added that the customary fêtes or amusements would begin in
about two months, and he would be very glad if I would stay and see
them; that he dressed now as a common man, but after that, I should
see him robed as a king. I told him I must go on early, to get to
Bornou before the rains. Mr. Houtson took this opportunity of
observing to him that he had been at the customs in Dahomey, and
inquired if the king of Yourriba put to death such a number of people
at his customs as at those of Dahomey. He shook his head,
shrugged up his shoulders, and exclaimed “No, no—no king of
Yourriba could sacrifice human beings; and that if he so
commanded, the king of Dahomey must also desist from that
practice; that he must obey him.”
Thursday, 26th.—This morning we had a sheep from the king, and
a hog and some plantains from one of his sons. In the evening I set
off five rockets, which astonished all and frightened away many. The
king was sitting under his verandah, and we waited on him to inquire
how he liked the rockets; he was quite delighted, and said they
should be kept for war.
Friday, 27th.—Employed in reducing the packages, and writing.
The morning dull and hazy. In the afternoon the king paid us a visit,
when we showed him some presents intended for the three principal
caboceers of the city. He said he did not know what to do or say for
our great kindness, as we had given him more things than he would
have got for the sale of one hundred slaves, and now we were giving
more to his caboceers; that, however, what he could do he would.
He said he had sent messengers in different directions to try to find a
safe path to the place where I wished to go; that while we were in his
dominions we were perfectly safe, but on leaving them he was sorry
to think we might be exposed to danger from the disturbed state of
the countries through which I must travel. He then said that the
Tappa, or Nyffé messengers, who had been here three years, were
in waiting to give us every information regarding the course of the
river that I might wish to ask him. They were accordingly called in,
and were certainly the most savage-looking knaves I ever saw; but
they either could not, or were afraid to give any the least account of
the river Quorra, and I therefore sent them off, after asking a few
questions. Indeed there seems a great unwillingness in both the king
and the people of this place to say any thing at all about the subject,
for what reason I cannot yet conjecture.
Saturday, 28th.—This morning I set out on horseback on a visit to
the three head caboceers, who dwell about three miles from our
house. We were received with much kindness and attention by all of
them under their respective verandahs, and surrounded by hundreds
of their wives, who all clapped their hands in token of welcome. They
severally presented us with goats, sheep, pigs, yams, eggs, honey,
and ducks, inviting us to drink country ale with them, and to make
merry; but I was very unwell, and anxious to get home. In the
evening we paid a visit of ceremony to the king, when I asked him to
allow Mr. Houtson and myself to go and look at the Quorra, and
return before closing my despatch for England. He replied, that he
heard what we said, and that we should go. I also asked for a
messenger to carry letters to Badagry in two days hence. He said he
would be ready.
Sunday, 29th.—Clear and cool. Very unwell all night with a bad
cold, pains in the limbs, and severe headache, with vomiting of bile;
took calomel. Richard also weak and unwell.
Monday, 30th.—Clear and fine. The harmattan seemingly
approaching its end. Better this morning. In the early part of the
evening we had no wind, and it is extremely hot.
Tuesday, 31st.—I have been very restless and unwell all night.
The king sent twice to inquire after my health yesterday, and wished
to come and see me; but I was too weak to sit up to receive him. The
messenger sent to open the path to Nyffé not yet returned. The king
called to see me this evening, but I was asleep; he insisted,
however, that Mr. Houtson should allow him to look at me with his
own eye, and taking the candle, he did so, and observed, that having
looked on me I should be quite well in the morning. Mr. Houtson
asked him for the loan of a horse, to take an airing in the morning.
This his majesty could not comprehend: what could a man want to
ride or walk for nothing? if he rode or walked, he ought to go and see
one of the caboceers, and he would get a present of a sheep, or a
pig, or some yams; that would be doing good; so he said he would
send a horse in the morning, and he must go and see some of his
caboceers, and he would send to let them know he was coming. The
pain in my head has fallen into my left eye, with inflammation and
acute pain, accompanied with a light delirium. Poor Pascoe very
unwell.
Wednesday, February 1.—Strong breezes. My eye a little better.
Pascoe much better. The king, agreeably to his promise, sent a
horse and two eunuchs to attend Mr. Houtson in his ride. He visited
one caboceer, and was about to return home, when the whole of the
party begged he would visit another, or all the caboceers would
make a palaver with this one. Mr. Houtson went accordingly to see
the other caboceers; he was received with great kindness and
attention, and came home with a supply of eggs, milk, honey, two
goats, a pig, two ducks, and plantains, &c. He objected to receiving
presents, but they told him the king’s friends could not come to their
houses, and go away empty-handed.
Sunday, 5th.—Morning clear, and a fresh breeze. In the afternoon
had a visit from his majesty. I asked him if the Nyffé messenger had
arrived. He said, no; that he must be dead, sick, or taken prisoner.
He said we could not go by the road of Nyffé, which was impassable
from the wars: what was my hurry to go? He was not yet tired of me;
he had many caboceers coming from the country to see me; he
wished to put every thing right on the roads for me before I set off;
that the king of England did not send me to him to run away again
directly; that he wished me much to wait and see the customs, for
then I should see him truly a king. I said I would do so with pleasure,
but that the rains would have set in by that time, and I should be
unable to go to Bornou. He asked what I was going to Bornou for.
“Did not the king of England send you to me alone?” “No,” said I, “he
sent me to you to procure me a passage to that country, where an
Englishman now resides who was left there when I returned from
thence.” I then told him I would consent to remain twelve days
longer, and if he did not by that time find me a passage, I would
return to England and say he would not allow me to proceed. He
now informed me that the messenger who arrived yesterday was
from one of his provinces called Yaru, five days distance; that it was
divided from the Youri by the Quorra; that he would send me by that
route, which was quite safe. I asked if I could not go and see the
Quorra before I departed from Katunga. He said no: the Fellatahs
had possession of the road. He gave me his gooro-nut box, carved
in the shape of a tortoise in ebony. I promised to let him have thirty
musquets, with powder and ball; on which he went away dancing,
tripped and fell, but was soon picked up by his ladies. He always
brings us some little present when he comes, and to-day he brought
us a bottle of honey, and some fruit called agra, about the size of a
pear, with a hard outer skin, four large black seeds, surrounded by a
pleasant acid pulp, like tamarinds, of a cream colour. My servant
Pascoe met in the market to-day some Fellatahs, who told him there
was no war in Nyffé; that the king was only afraid of the Fellatahs;
that the Fellatahs of Raka had taken nine Yourribanis, who had been
found in a suspicious place, but were going to return them here on
the morrow. Raka is only one day’s journey north-north-east from
Katunga.
Monday, 6th.—In the evening, at the request of the king, I again
set off five rockets, one of which having too low an elevation ran
along the ground, but fortunately only set fire to some grass. We
afterwards went and saw the king, who, with his ladies and principal
men, was sitting outside under the verandah to see the rockets. He
presented us with gooro nuts, and said he would come and see us in
the morning.
Tuesday, 7th.—In the middle of the day the king visited me, and
brought a bottle of honey and two cock fowls. He began joking me as
to what I was about to give him. I said I had nothing to give him.
Says he, “I ask you to give me one of your servants.” “I can’t do
that,” says I; “they are free men as well as myself.” “What,” says he,
“no slaves in England!” “No,” says I, “as soon as a slave sets his feet
in England he is free.” “Then,” says he, “as you must go, either Mr.
Houtson or Richard must stop with me. I must have one.” After a
good deal of conversation of this kind I asked him to fix a day for our
departure. He artfully shifted the subject of conversation to that of
women. Would I not like a wife? he would give me one. Did he not
give us plenty to eat, or did he not use me well? “All very true, and
very good,” says I, “but I am not like a black man, who has no book
to write. I must know the day on which I am to go, as I must have all
my books ready for the king of England. Every thing I give away is in
that book, every thing I get, and every thing I say.” All my talk would
not make him fix a day, but he said I should have a servant of his to
the king of Youri; that that road was safe. I would go in four days to
Yara in Bamba, which was tributary to him; there I would cross the
river Moussa, which ran into the Quorra, three days distance; that
the Moussa came from the north-west, and in it were plenty of
hippopotami. He is still particularly shy of giving any information
about the Quorra, of which, perhaps, he has none. At one time he
says it runs into the sea between Jaboo and Benin, and at another
that it passes Benin; that the Fellatahs are in possession of Raka,
only a day’s journey north-north-east, and of all the country between
it and the Quorra: he therefore cannot contrive to get me thither. He
now shifted the subject of conversation; told me he did not know how
many wives, or how many children he had got, but he was sure that
his wives alone, hand to hand, would reach from hence to Jannah.
He sent for one of his daughters, whom he had given as wife to
Abaco, the messenger. His daughters are allowed to take any one
they may choose, either as a husband or lover; but it is death to
touch any of the king’s wives. The son, at the father’s death, takes all
the widows to maintain. The king had his skin rubbed over with the
powder of a species of red wood, ground very fine, and made like a
paste; it is used by all classes. The wood is brought from Waree and
Benin. We gave him a flask of rum on his leaving us, and he
promised to give me some of the blue stone of which his beads are
made. He says it comes from a country between this and Benin.
They are not glass, as I at first supposed.
Thursday, 9th.—This evening I had to take the eunuch to task
about our provisions: he had been dealing us out too small a share,
and pocketing the rest. He pretended to be in a great rage, and even
the milk is now bad.
Friday, 10th.—Moderate breezes and clear. A number of
caboceers from distant provinces arrived to-day, and we had nothing
but drumming, and whistling, the whole day. The king sent us an
invitation to see them, and we went in the afternoon. We found the
king seated in an old easy chair covered with crimson damask. The
caboceers at some distance in front, facing him, dressed in leopard
skin robes, their heads well dusted, and also their cheeks, by
rubbing their faces in the dust while making their prostrations. It is
the court etiquette here to appear in a loose cloth, tied under one
arm; part over the other shoulder, and hanging down to the feet in a
graceful manner: but no tobes, no beads, no coral, or grandeur of
any kind, must appear but on the king alone. The cane I made him a
present of holds, on all occasions, a conspicuous place: when he
walks, he carries it, and when he sits, it is stuck in the ground at
some distance before him. He presented us with gooro nuts, and
asked me to fire off some rockets to-night.
The caboceers from the country were attended by their bowmen.
They are required to wait upon, and first to prostrate themselves
before, the chief eunuch, with dust on their heads. When any one
speaks to the king, he must do it stretched at full length on the
ground, and it must be said to him through the eunuch, who is also
prostrated by his side. When equals meet, they kneel on one knee;
women kneel on both knees, the elbows resting on the ground.
Saturday, 11th.—More caboceers arrive to-day, attended by their
wild-looking followers, armed with swords, bows, and arrows; they
also, covered with dust and sweat, went through their prostrations
before the fat eunuch; the attendants dancing in a circle, while
occasionally one came out, and danced a movement in the minuet
style; in doing which, he would frequently throw a somerset equally
as expert as old Grimaldi himself in his best times. I sat for an hour
and a half, during all which time the prostrations were continued
without intermission, accompanied with the dancing and tumbling,
without regard to the intense heat of the sun. I concluded that they
were practising before the eunuch, in order to be perfect when they
appeared before the king. They were dressed in leopard skin robes,
hung round with tassels and chains. At last the prostrations were
completed, and the eunuch sent for several jars of palm wine. The
caboceers were admitted to drink theirs in the eunuch’s house, but
the attendants drank their share under a tree. In the afternoon, the
king sent for us to see him, but I was too unwell to go, and desired
Mr. Houtson and Richard to attend him.
Monday, 13th.—This morning, our friend and guardian, the fat
eunuch, was drunk; when in that state, he begs every thing he sees.
He got Mr. Houtson and myself into his house to see him dance; but
independent of his want of steadiness, he was the most clumsy and
unwieldy performer I ever saw. He begged we would also dine with
him, but I complained of illness, and Houtson ran off. He followed
and made Mr. Houtson hand out the flask, which, without waiting for
a glass, he put to his mouth, and drank upwards of a pint of raw rum,
without drawing his breath. He said, he had drank two quarts to-day
already, and given away a small cask: that rum was good, and made
him fat.
The people of Katunga are fond of ornamenting their doors, and
the posts which support their verandahs, with carvings; and they
have also statues or figures of men and women, standing in their
court yards. The figures carved on their posts and doors are various;
but principally of the boa snake, with a hog or antelope in his mouth;
frequently men taking slaves, and sometimes a man on horseback
leading slaves.
Their manner of burying the dead is to dig a deep narrow hole,
into which the corpse is put in a sitting posture, the elbows between
the two knees: a poor person is buried without any ceremony; a rich
man has guns fired, and rum drank over his grave, and in his house
by his friends and retainers. When a king of Yourriba dies, the
caboceer of Jannah, three other head caboceers, four women, and a
great many favourite slaves and others, are obliged to swallow
poison, given by fetishmen, in a parrot’s egg: should this not take
effect, the person is provided with a rope to hang himself in his own
house. No public sacrifices are used, at least no human sacrifices,
and no one was allowed to die at the death of the last king, as he did
not die a natural death; having been murdered by one of his own
sons: not the present king. Wives are bought; and according to the
circumstances of the bridegroom, so is the price. Three days after
the bargain, he and his friends go and bring the wife to his own
house, when the pitto, or country beer, is sent about freely amongst
the guests.
In the afternoon we waited on the king. There is a pleasant walk
through a large enclosed park at the foot of the hills, between the
house of the king and that of his wives, enclosed by a clay wall.
Some parts of the park are planted with corn, yams, &c. and others
studded with beautiful shady trees. The king was sitting under the
shade of one of the trees. I observed to him that I had been here
twenty-four days, and was anxious to go on my journey, as the rains
were about to set in. He asked if all the white men were going. I said
“Only myself and my servants.” As I knew him to be skilful in evasive
answers, and always to have one ready at hand, I said “Fix a day.”
His reply was, “Every one would say, the white man came to see the
king of Yourriba, and brought him large presents, and requested him
to give them a good passage to where they wanted to go; he gave
them a bad path; they were robbed and killed: all people would say
that the king of Yourriba did not do good to white man.” He had been
busy these last four days with his people, but he had sent a
messenger to get a good path. I asked him positively to fix a day, as
I could not put off any longer. After consulting with his people, he
said, “Nine days.” I said, “Well—I shall remain nine days:” without
saying one word that I had every thing ready to go.
Foo-foo, the common food of the rich and poor, is of two kinds—
white and black: the white is merely boiled yams beaten into a paste
with water, and sold in balls of about a pound weight each. The black
is made as follows:—The yam is first parboiled, then cut into small
pieces and exposed to the rays of the sun till quite dry; in this state it
is pounded in a large wooden mortar into a flour, and sifted again
and again until it is as fine as possible. The flour will keep in this way
about six months. When wanted for use, boiling water is poured over
it, and stirred round until completely mixed and of a proper thickness;
and when so done it is, like the other, made into balls of about a
pound weight each. The natives eat it with soup, gravy, or palm oil;
or without any other thing.
Wednesday, 15th.—At 3 P.M. we had a messenger from Jannah,
who brought letters; one from Captain Clavering of the Redwing, with
half a dozen bottles of porter and half a dozen of wine; the other two
half dozens having either been broken or drunk on the road. The
supply was as welcome as unexpected, and proved to us how very
little trouble and expense were required to keep up the
communication. By the letters of Captain Clavering, it appears that
the trunks of the late Captain Pearce and Dr. Morrison had arrived
safe.
Thursday, 16th.—Morning cool and cloudy. We had an early visit
from the king, who was anxious to have a bottle of our porter, which I
could not well refuse. He also begged my looking glass, and one of
the tin boxes. In my turn I told him, that since we had finished our
bullock that he gave us, we had nothing to eat. He replied, we must
go to the caboceers. I told him I would do no such thing; that we did
not come here to beg. He said I was mistaken; it was not begging;
they were all his slaves, and what they possessed belonged to him. I
requested, however, that he should send orders to them direct from
himself. After a little time he left us: but the eunuch remained; and
showing symptoms of sauciness I turned him out of the house.
Friday, 17th.—Morning clear and cool, with fresh harmattan
breezes. A great number of people arrived to-day from different parts
of the country to pay their annual custom and visit to the king.
Saturday, 18th.—Morning clear and cold; a strong harmattan still
blowing. The religion of the people of Yourriba, as far as I could
comprehend it, consists in the worship of one God, to whom they
offer sacrifices of horses, cows, sheep, goats, and fowls. At the
yearly feast all these animals are sacrificed at the fetish house, in
which a little of the blood is spilt on the ground. The whole of them
are then cooked; and the king and all the people, men and women,
attending, partake of the meat, which they are said to eat in a state
of nakedness, and in company, drinking at the same time copiously
of country ale, or pitto; but it is also said, that the least attempt at
indecency would be punished with death.
It is stated, moreover, that it depends on the will of the fetish man
or priest whether a human being, or a cow, or other animal is to be
sacrificed. If a human being, it is always a criminal, and only one.
The usual spot where the feast takes place is in a large open field
before the king’s house, under wide spreading trees, where there are
two or three fetish houses. This account I had from a native of
Bornou, a Mahometan, and a slave to the caboceer of Jannah.
Monday, 20th.—The morning cool. The harmattan still continues
blowing. Our supply of provisions has of late been rather scanty,
owing to the avarice of the fat eunuch, our guardian, who pockets as
much as he can out of what the king sends us. I have threatened him
two or three times, but I believe he trusts to my patient and easy
disposition. Complaining to the king would get him a beating, though
he is a favourite, and an useful person, well-skilled as a war-captain,
and guardian of the king’s women.
Tuesday, 21st.—Cool and clear. Harmattan continues. A number
of caboceers of the different towns came in to-day with their forces,
and the king sent for us to come and see them. We went accordingly,
and saw about twenty of these dependents, in all their dirt and
debasement, who vied with each other which could have most dust,
and who could kiss the ground with the greatest fervour. They were
stretched at full length on their bare bellies; no cloth being allowed
on this occasion over the shoulder, the body being required to be
bare as far as the waist. Old Pascoe calls them the sand-eaters.
After our usual compliments to the king, and shaking hands with the
caboceers of Eyeo and the sand-eaters, we returned; the king
promising to visit us at our house.
At 4 P.M. he came, attended by his women and our fat guardian.
The women he left outside, except two; one of whom attends upon
him on all occasions bearing a handsome carved gourd, having a
small hole covered with a clean white cloth, to hold his Majesty’s
spittle, when he is inclined to throw it away; the other with a white
pot, used with us as a chamber utensil, containing his gooro nuts,
since he made me a present of his black ebony box, carved in the
shape of a tortoise, which he used for that purpose. After an end had
been put to our complimentary inquiries after his majesty’s good
health, I observed that the time was now come, within a few days
only, that he had appointed for my departure. He said, that the
messenger he had sent to Youri was returned; that the road was
perfectly safe; that he would have me passed from one king to
another; but that by the way of Nyffé he would not ensure my safety;
and that if he suffered me to go where there was danger, it would
cause a reflection on him. I thanked him for his kindness, and said
that whatever he did was right. He then said that his messenger, and
that of the caboceer of Yarro, would attend me to Youri. I thought this
a proper time to hint a gentle complaint against our fat guardian, for
having for some time past appropriated our provisions to his own
use. The old rogue swore through thick and thin that he had given us
every thing, even some goats which I had actually purchased at the
market, but which he swore he had supplied himself. I told the king it
was of no use talking against a rogue like his eunuch, therefore I
should hold my tongue. As the king never comes to us empty-
handed, he brought us a Muscovy duck, and a bag of rice; the last a
scarce article, and not to be had in the market.
Wednesday, 22d.—Cold morning. Harmattan still continues. More
caboceers came in yesterday, with their attendants. They waited on
us this morning, and we observed that they were well provided with
dust, as they had been to wait on the king early; it being the etiquette
of Yourriba to hold a levee twice a-day, at six in the morning, and at
two in the afternoon.
It is the custom, during the time that the caboceers from the
different towns remain on their visit to the king, to act plays or
pantomimes, or whatever they may be called. I shall attempt a
description of the one I saw to-day. The place chosen for this
pastime is the king’s park, fronting the principal door where his
majesty usually sits. A fetish house occupies the left side; to the
south are two very romantic and large blocks of granite, by the side
of which is an old withered tree. On the east are some beautiful
shady trees; and on the north his majesty’s house, from whence he
views the scene. In the centre are two beautiful clumps of trees; in
one of which is a tall fan-palm, overlooking the whole area, a space
that may include some seven or eight hundred yards square. Under

(FREE PDF Sample) Microcalorimetry of Biological Molecules Methods and Protocols Eric Ennifar Ebooks

Uploaded by

Copyright:

Available Formats

(FREE PDF Sample) Microcalorimetry of Biological Molecules Methods and Protocols Eric Ennifar Ebooks

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

(FREE PDF Sample) Microcalorimetry of Biological Molecules Methods and Protocols Eric Ennifar Ebooks

Uploaded by

Copyright:

Available Formats

Full download test bank at ebook textbookfull.

CLICK LINK TO DOWLOAD

Nucleic Acid Crystallography Methods and Protocols 1st

Zebrafish Methods and Protocols Koichi Kawakami

SNAREs: Methods and Protocols Rutilio Fratti

Epitranscriptomics: Methods and Protocols Narendra

Metalloproteins: Methods and Protocols Yilin Hu

Nanotoxicity: Methods and Protocols Qunwei Zhang

Autoantibodies: Methods and Protocols Gunnar Houen

Oligodendrocytes: Methods and Protocols David Lyons

Eric Ennifar Editor

For further volumes:

Methods and Protocols

ISSN 1064-3745 ISSN 1940-6029 (electronic)

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Although structural biology is key to elucidate molecular architecture of biological mole-

Strasbourg, France Eric Ennifar

PART I DIFFERENTIAL SCANNING CALORIMETRY

PART II ISOTHERMAL TITRATION CALORIMETRY

5 Intrinsic Thermodynamics of Protein-Ligand Binding by Isothermal

12 Characterization of Microtubule-Associated Proteins (MAPs)

PART III ADVANCED DATA PROCESSING AND KINETICS

13 Analysis of Isothermal Titration Calorimetry Data for Complex

ADRIAN VELAZQUEZ-CAMPOY  Institute of Biocomputation and Physics of Complex Systems

Differential Scanning Calorimetry

The Contribution of Differential Scanning Calorimetry

Key words Membrane-active peptides, Peptide/lipid interaction, Differential scanning calorimetry,

Membrane lipid properties (fluidity, thickness, ordering, etc.) vary

(Pβ0 ). In this intermediate phase, the bilayer is still highly ordered

two phases is called TH and occurs at higher temperatures than Tm

membrane fluidity, cooperativity of the transition, and others that

proportion of chloroform and methanol can be adjusted

1 C/min with a delay of 10–15 min between sequential scans

thermal changes to be observed by DSC at the low concentra-

perform the deconvolution of the thermogram and identify the

the line, at ΔH ¼ 0 one can obtain the number of lipid molecules

1. Ideal buffers for DSC measurements should undergo minimal

absolutely needs to be considered is that the buffer composi-

tendency to greatly broaden the transition, so again higher lipid

Protocols of IATC, DSC, and PPC: The Multistate Structural

Calorimetry is the strongest method for evaluating the thermody-

an effective technique for evaluating the thermal expansion coeffi-

2.2 Preparation 1. Blank solution: 50 mM acetate buffer at pH 4.1. Prepare 2 L of

3.1.2 pH Measurement 1. Calibration of pH meter: Perform the three-point calibration

RESIDENCE AT EYEO, OR KATUNGA, THE CAPITAL OF YOURIBA.

Tuesday, January 24th.—Early this morning the king paid us a

You might also like

ADRIAN VELAZQUEZ-CAMPOY Institute of Biocomputation and Physics of Complex Systems