PROTEIN CHEMISTRY | BIOCHEM A |Sources: Dra. Santos Lecture; LEA THERESE R.

PACIS’ trans

CHEMISTRY OF AMINO ACIDS, PEPTIDES AND PROTEINS 6. Control of Growth and Differentiation
REMEDIOS P. SANTOS, MD Ex. Repressor proteins are important control elements that
silence a specific DNA of a cell. This is essential for the orderly
I. PROTEINS growth and differentiation of cells.
Important in gene expression.
- third major nutrient that the body needs (carbohydrates,
lipids); 7. Cell Signaling
- proteins are the most important -- we cannot live without Ex: membrane receptors such as insulin receptor
proteins.
- complex, organic nitrogenous substances with very high molecular 8. Hormones
weight Protein hormones include Insulin, Thyrotropin,
- Found in all plant and animal cells Somatotropin (Growth Hormone), Luteinizing Hormone,
- Consists largely or entirely of alpha-amino acids united in peptide and Follicle Stimulating Hormone (FSH)
linkages
AMINO ACIDS = basic unit by which proteins are made of 9. Proteins are one of the major components of biological
- They are joined together through the PEPTIDE BONDS membranes (other one is lipid)
- There are 21 amino acids
- “alphabets of proteins”  CLASSIFICATION OF PROTEINS:
 Comparable to the letters in the alphabet, with amino acids –
the amount/number or the sequence of amino acids could A. Classification Based on Composition, Physical and
vary and would come up with proteins. Chemical Properties of the Protein
 Sickle Cell Hemoglobin VS Normal Hemoglobin = Differ in
one amino acid; 1. SIMPLE PROTEINS = made up of AMINO ACIDS ONLY
 This goes to show that the difference between a normal
protein and an abnormal protein could just be because of  Albumin
one amino acid. - Soluble in water & dilute aqueous salt solution
- Derived from the Greek word “proteios” = “primary” or “holding - Heat coagulable
first place” or “of first importance”
- proteins are among the most important substances which make up  Globulin
the human body - Insoluble in water
- G. J. Mulder = Dutch chemist coined the word “protein” in 1893 - Soluble in aqueous solution
- Heat coagulable
 FUNCTIONS OF PROTEINS:
 Glutelin
1. Enzymatic Catalysts - Soluble in dilute acids and alkalies
Ex: enzymes involved in different metabolic pathways. - Heat coagulable
ORYZENIN
Enzymes, which are important in the different metabolic  Protein found in rice
processes in the body, are protein in nature.  Incomplete Protein
- needed to digest our food; COMPLETE PROTEIN VS INCOMPLETE PROTEIN
 to get the nutrients from the food that you eat; Complete Protein Incomplete Protein
 If we cannot digest our food, we become malnourished,  Contains all the  Lacks one or more of the
we die. essential amino acids essential amino acids
- needed in the different metabolic pathways (Kreb’s Cycle,  Can sustain life by (even if it just lacks one
glycolysis) itself amino acid)
 No enzymes = defective pathways = no energy Ex: Protein found in milk,  Cannot sustain life by
meat, and eggs itself
2. Transport and Storage
Ex: hemoglobin  Prolamine
HEMOGLOBIN transports oxygen. - alcohol-soluble protein
ZEIN = Protein found in corn
3. Coordinated Motion
Ex. actin and myosin which are involved in muscle
 Albuminoid or scleroprotein
contraction - least soluble
Mechanical Support Protein found in exoskeletal structures HAIR & NAILS
Ex: collagen and keratin
Collagen  Histone
- most abundant protein in the body; - soluble in water, dilute acid and alkali;
- no collagen, no support to the different tracts in the body; - basic protein (Contains a lot of basic amino acids)
including of blood vessel walls - found in combination with DNA
- VITAMIN C is important in the synthesis of good collagen.
 Protamine
4. Immune Protection  soluble in water, dilute ammonia, acid and alkali;
Ex. Antibodies derived from gamma globulins  simplest protein; basic;
 found in spermatozoa
5. Generation and Transmission of Nerve Impulses
Ex: neurotransmitters derived from different amino 2. CONJUGATED PROTEINS
= made up of AMINO ACIDS + OTHER SUBSTANCE
Serotonin
(PROSTHETIC GROUP)
- derived from Tryptophan
- neurotransmitter, vasoconstrictor, and a GIT regulator
(regulates GI tract motility).

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 PROTEIN CHEMISTRY | BIOCHEM A |Sources: Dra. Santos Lecture; LEA THERESE R. PACIS’ trans

 Nucleoprotein 4. STRUCTURAL PROTEINS - they are said to function in

 has NUCLEIC ACIDS as prosthetic group “brick and mortar” roles.
 Glycoprotein and Mucoprotein Ex. collagen, proteoglycans, elastin
 has CARBOHYDRATES as prosthetic group
 Phosphoprotein 5. PROTECTIVE PROTEINS - blood clotting factors,
 has PHOSPHORIC ACID residues as prosthetic group immunoglobulins, interferon
Ex. CASEIN – protein found in milk
 Chromoprotein 6. TRANSPORT PROTEINS - hemoglobin, plasma
 contains prosthetic groups that give COLOR lipoproteins, transferrin
“chromo” – means color
7. CONTRACTILE or MOTILE PROTEINS - actin, myosin
Ex: HEMOGLOBIN – “heme”, means blood(red)
 Lipoproteins
Some proteins have functions that are rather exotic and not easily
 contains LIPIDS
classified.
Example: LDL, HDL, Chylomicrons
Examples of these proteins are:
 Metalloproteins
1. MONELLIN
 contains METALS
 Protein of an African plant which has an intensely sweet taste
Example: Insulin, Cytochrome
 Being studied as a non-fattening, nontoxic food sweetener for
human use
3. DERIVED PROTEINS
2. “ANTI-FREEZE” PROTEIN
a. Primary Derived
 Found in the blood plasma of some Antarctic fish which
b. Secondary Derived
protects their blood from freezing
3. RESILIN
B. Classification Based on the Shape and Certain Physical  Where wing hinges of some insects are made of
Characteristics of the protein:  Has nearly perfect elastic properties

1. FIBROUS PROTEINS = elongated; look like ropes II. AMINO ACIDS (AA)
 Tough
 Insoluble in water  group of relatively simple building-block molecules from which
 Arranged around a single axis to form a fiber proteins are built.
 Involved in structural functions  All proteins, whether from the most ancient lines of bacteria or
Ex. Collagen and Keratin from the highest forms of life, are constructed from the same basic
 form the matrix of bone and ligaments set of 21 amino acids (inc. Selenocysteine), covalently linked in
 provide structural and elasticity to organs and the characteristic sequences.
vascular system  each AA has a distinctive side chain responsible for its chemical
 where skin derives its strength and flexibility individuality, thus regarded as “alphabet of protein structure”

 Collagen - it is the most abundant of the fibrous proteins Again, AAs are the BASIC UNIT by which proteins are made of.
that constitutes more than 25% of the protein mass in the Whatever is the characteristic of the amino acid found in the
human body. protein will have an impact on the function of the protein.
 Keratin - it is the chief structural component of hair, scales,
horns, wool, nails and feathers. It is also the principal STANDARD, PRIMARY, OR NORMAL AMINO ACIDS
component of the tough armor of the tortoise (turtle).  20 amino acids of proteins; Referred to as such to distinguish
them from other kinds of amino acids present in living organisms
2. GLOBULAR PROTEINS = circular but not in proteins
 Involved in mobile and dynamic functions NONSTANDARD AMINO ACIDS
Ex. Enzymes, Hemoglobin, Plasma Proteins  consists of amino acid residues that have been chemically
Plasma proteins – albumin, globulin, fibrinogen, hemoglobin modified after they have been incorporated into a polypeptide
and amino acids that occur in living organisms but are not found
The shape of the protein is based on its function; or function in proteins
dictates the shape. Ex.
Hemoglobin  Conversion of peptidyl proline and lysine to 4-hydroxyproline
 Rounded and 5-hydroxylsine
 Found inside the red blood cells - it follows the shape of  Conversion of peptidyl glutamate to gamma carboxyglutamate
the RBC.  Methylation, formation, acetylation, and phosphorylation of
certain aminoacyl residues
*These modifications extend the biologic diversity of proteins by
Molecular Chaperones altering their solubility, stability, and interaction with other
 group of substances that will guide a particular peptide chain to proteins.
assume the shape that is in consonant with its function; or
 simply, guides the shape of peptide chain
 FUNCTIONS OF AMINO ACIDS:
C. Classification Based on Biologic Functions: 1. Building blocks of proteins – “alphabet”; primary function
2. AA are precursors of many important substances, a variety
1. ENZYMES - dehydrogenases, kinases, etc. of complex, nitrogen-containing molecules:
Enzymes are called CATALYTIC PROTEINS because they are Ex.
involved in catalysis.

2. STORAGE PROTEINS - ferritin, myoglobin

3. REGULATORY PROTEINS - DNA-binding proteins, peptide

hormones

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 PROTEIN CHEMISTRY | BIOCHEM A |Sources: Dra. Santos Lecture; LEA THERESE R. PACIS’ trans

a) GLYCINE – heme, purines, creatinine, glutathione, hippuric Proline and Lysine have been modified by hydroxylation to
acid produce Hydroxyproline and Hydroxylysine.
 HEME = Glycine + Succinyl-CoA (intermediate of Krebs Cycle)  important components of Collagen.
 PURINES (Picture Below: Adenine)
 N3 and N9 = comes from Structure of Collagen:
Glutamine  Made up of Glycine (most abundant), and X and Y AA
 N1 = comes from Aspartic Acid  Hydroxyproline, Hydroxylysine, or Proline (usually X&Ys) -
 C4, C5, N7 = comes from Glycine are responsible for the tensile strength of Collagen; makes
 Purines are found in DNA. collagen strong and hard.
 No amino acids = No purines =  To form Hydroxylysine and Hydroxyproline, you need to
No DNA = No cell growth, have VITAMIN C (hydroxylase proline and lysine) = NO VIT
multiplication, maturation = No LIFE C = Collagen is abnormal/not strong
 CREATININE = GlAM (Glycine, Arginine, Methionine)
 GLUTATHIONE = GCG (Glycine, Cysteine, Glutamic Acid)  BLOOD VESSELS
If collagen is abnormal, your blood vessels become fragile.
b) GLUTAMIC ACID – GABA (gamma-aminobutyric acid) May cause vessels to break, Ex. Of Vit C deficiency: NOSE
GABA - our natural tranquilizers. BLEEDING (EPISTAXIS).
No GABA = convulsive seizures.
Glutamic Acid + Vit B6 (co-enzyme) = GABA b. N-METHYLLYSINE - found in myosin
c. GAMMACARBOXYGLUTAMIC ACID - component of
c) PHENYLALANINE and TYROSINE – melanin, dopamine, prothrombin
thyroxine, epinephrine and norepinephrine Gammacarboxylglutamic Acid
Phenylalanine + oxygen = Tyrosine =  is found in Prothrombin (important for blood coagulation)
 THYROXINE = Thyroid Hormone  a special type of Glutamic Acid that has undergone
 MELANIN = Pigment responsible for your complexion gammacarboxylation
 EPINEPHRINE = important hormone for the control of your  VITAMIN K = Necessary for gammacarboxylation =
blood sugar level important for the synthesis of Prothrombin
 Stimulates Glycogenolysis and Gluconeogenesis =  No Prothrombin = bleed to death
Increases Blood Sugar Levels
 Hyperglycemic Hormone d. DESMOSINE - derivative of lysine, found in elastin
 NOREPINEPHRINE e. N-ACETYLLYSINE - found in histones that are associated
with chromosomes.
d) TRYPTOPHAN – niacin, serotonin, indole and skatole,
melatonin 5. Phosphorylation and dephosphorylation of Serine,
Threonine and Tyrosine play major roles in the signal
 NIACIN = Vitamin B3
transduction pathways by which cells communicate with and
 Pellagra - Niacin Deficiency;
respond to their environment.
 3Ds: Dermatitis, Diarrhea, and Dementia
 SEROTONIN = neurotransmitter; vasoconstrictor
Important for the activation and inactivation of enzymes.
 MELATONIN = hormone that induces sleep
 Substance found in sleeping tablets (Sleepasil)
Phosphorylation – addition of phosphate
 INDOLE AND SKATOLE = odor of the stool
Dephosphorylation – removal of phosphate

e) HISTIDINE – Histamine Hydroxyl (-OH) containing AA

HISTAMINE - substance released when you have allergies.  AA that can accept phosphate (phosphorylation)
Common manifestation: Skin rash  SERINE, THREONINE, and TYROSINE
Others:
 Increased gastric acid secretion = vomiting, diarrhea = You can attach Phosphate to –OH  Becomes CH2OPhosphate
dehydration (electrolyte imbalance)
 Bronchospasm = difficulty in breathing Ex.
 Severe vasodilatation = BP drops = anaphylactic shock  GLYCOGEN PHOSPHORYLASE
 Usually injected with something you’re allergic to.  enzyme activated by Phosphorylation
 for Glycogenolysis (breakdown of glycogen)
f) LYSINE and METHIONINE – carnitine  GLYCOGEN SYNTHASE
 CARNITINE – for weight reduction, with exercise. - enzyme activated by Dephosphorylation
- for Glycogenesis (glycogen synthesis)
-
3. Source of energy
Proteins = source of energy, just like Carbohydrates and Lipids 6. L--amino acids in low molecular weight peptides play additional
 1 gram of Carbohydrates = 4 calories roles as hormones.
 1 gram of Fats = 9 calories 7. Both D- and L--amino acids are present in polypeptide
 1 gram of Protein = 4 calories antibiotics elaborated by microorganisms.
Ex. Bacitracin, Gramicidin A
8. Several -amino acids or their derivatives act as chemical
4. Special amino acids as components of certain types of messengers.
proteins - these amino acids arise by “post-translational” Ex. Glycine, GABA, Serotonin and Melatonin - neurotransmitters
modifications of an amino acid that has previously been 9. Hormones can be derived from amino acids: Thyroxine,
incorporated into a peptide or protein. Epinephrine, and Norepinephrine from Phenylalanine and
Thyrosine
a. HYDROXYPROLINE and HYDROXYLYSINE 10. Several standard and non-standard amino acids act as
 present in collagen metabolic intermediates.
 Arginine, Citrulline, Ornithine - components of the Urea Cycle

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 PROTEIN CHEMISTRY | BIOCHEM A |Sources: Dra. Santos Lecture; LEA THERESE R. PACIS’ trans

 BASIC STRUCTURE OF AA:  AMINO GROUP

 can accept a proton/ Hydrogen ion (H+) = to become NH3+
Each amino acid has a central carbon, called the ALPHA-  BASE - proton acceptor (Bronsted-Lowry Definition)
CARBON, to which 4 different groups are attached:  the BASIC part of the AA
1) a basic amino group (-NH2)
2) an acidic carboxyl group (-COOH)  CARBOXYL GROUP
3) a hydrogen atom (-H)  can dissociate into a carboxylate ion(-COO-) + proton(H+)
4) a distinctive side chain (-R)  ACID - proton donor (Bronsted-Lowry Definition)
 the ACIDIC part of the AA
C = ALPHA CARBON
3 Reactive Parts: AMPHOTERIC PROPERTY
1. Amino Group (-NH2)  AA can act as a base and an acid at the same time
2. Carboxyl Group (-COOH)  has both a basic part and an acid part
3. Side Chain (-R)  All AAs are amphoteric.

FORMULA OF AMINO ACIDS:

 All AAs are the same as to:
 Alpha Carbon
 Amino Group
 Carboxyl Group
 Hydrogen Group

 R-Group/(-R) (side-chain)
 R-Group distinguishes one AA from another AA.
 Gives specific characteristic of the AA
The common amino acids are known as alpha-amino acids  Attached to -carbon = center, not the carboxyl group
(-amino acids) because they have a primary amino group and a  the only part of the AA that is changing
carboxylic acid group as substituents attached to the alpha-carbon Ex. -R= CH3 = Alanine; -R= CH2OH = Serine; -R= H = Glycine
atom.

EXCEPTION:
PROLINE, has a secondary amino group
(-NH-), referred to as -IMINO ACID, since
its nitrogen is bonded to the -carbon and the
side chain group(-R); Gives a polypeptide
important structural features, due to its *Memorize the formula of AAs by memorizing the changing Rgroups
structure.
 CLASSIFICATION OF AMINO ACIDS BASED ON POLARITY
ALPHA-AMINO ACID - both the amino group and carboxyl
group are attached to the alpha-carbon A. Amino acids with NONPOLAR or HYDROPHOBIC RGROUPS

BETA-AMINO ACID - amino group is attached to the beta-  NEUTRAL AMINO ACIDS, containing hydrocarbon R groups
carbon; not found in the body  NEUTRAL = R groups do not bear positive or negative charges
 interact poorly with water = important role maintaining the 3D
WHY DO YOU CALL IT L-ALPHA-AMINO ACIDS? structure of proteins
 different from Levorotatory (Optical isomerism) - small letter L (l)
or (-) sign  Types of Hydrocarbon Side Chains
 Same concept with D-Glucose and L-Glucose > position of the a. Aromatic
–OH at the penultimate carbon  Phenylalanine – Benzene ring
 -OH on the LEFT of the penultimate carbon = L-Glucose  Tryptophan – Indole ring; rgroup - indole group
 -OH on the RIGHT of the penultimate carbon = D-Glucose *Phe and Trp contain aromatic ring structure.
*Tyrosine, another aromatic AA, polar, also called hydroxyl
 in AAs, it depends on the position of the amino group relative to phenylalanine.
the -carbon b. Aliphatic
 Amino group on the LEFT of -carbon = L-Amino Acid  Glycine
 Amino group on the RIGHT of -carbon = D-Amino Acid  Alanine
 Valine
 Majority of AAs in the body are L-Amino Acids; only 2 are  Leucine Branched-Chain Amino Acids
D-Amino Acids: D-ASPARTATE and D-SERINE - in the brain  Isoleucine
 Majority of D-AAs can be found in bacterial cell walls.  Methionine
 Proline
 SIDE CHAINS (-R) - functional groups that are the major
determinants of the conformation and function of proteins and Branched-chain amino acids
electrical charge on the molecule. - R group branches
 thus, properties of each AA are dependent on its (-R). - Also called bulky Rgroup
 to understand methods of analysis, purification, and
identification of proteins, knowledge of the properties of these NOTE on Solubility;
side chains is necessary. The Longer the Rgroup = the less soluble
 Amino acids with CHARGED, POLAR OR HYDROPHILIC Val Rgroup is shorter than Leu and Ile; Thus, Val is more
SIDE CHAINS - usually exposed on the surfaces of proteins. soluble than the other 2.
 NONPOLAR HYDROPHOBIC RESIDUES - usually buried in
the hydrophobic interior of a protein, out of contact with water.

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 PROTEIN CHEMISTRY | BIOCHEM A |Sources: Dra. Santos Lecture; LEA THERESE R. PACIS’ trans

 Glycine – simplest (-R = H) and smallest AA; B. Amino acids with UNCHARGED POLAR R GROUPS
 it can be accommodated in places inaccessible to other AA  has functional groups capable of hydrogen bonding = can easily
(particularly where peptides bend sharply) interact with water
CHIRAL CARBON 1. Hydroxyl-containing amino acids
 carbon at the center of AAs; - Serine, Threonine, Tyrosine
 carbon where 4 different groups are attached - sites of phosphorylation reactions;
 ASYMMETRIC CARBON - site of glycosylation reaction
 capable of optical rotation (Dextrolevotatory Serine, Threonine, Tyrosine
or Levotatory) - contain polar hydroxyl groups = important factor in protein
EXEMPTION: GLYCINE structure.
(R group = H = thus, only 3 different groups Serine, Threonine -OH groups = points for attaching
= no chiral carbon) carbohydrates

 Proline – the “helix breaker”; also called an IMINO ACID 2. Amide-containing amino acids
Proline’s R-group is attached to the -carbon  Glutamine - derivative of Glutamate; primary source of
and to the amino group, giving its rigid and urinary ammonia.
cyclic appearance and structure.  Asparagine - derivative of Aspartate
 important in the detoxification of ammonia
3. Sulfur-containing
 Cysteine - Thiol group; polar because of its sulfhydryl group
 Methionine – source of SAM (S-adenosylmethionine), the
active methyl donor; sulfur containing

C. Amino acids with POSITIVELY CHARGED R GROUPS

(BASIC)
 Lysine
 Arginine
 R group = GUANIDO or GUANIDINIUM GROUP
 Histidine
 R group = IMIDAZOLE GROUP
(pK of its imidazole proton permits it to function at neutral
 NEUTRAL AMINO ACID pH as either a base or an acid catalyst)
 if there are no additional amino group and carboxyl group at  responsible for the buffering capacity of hemoglobin.
the sides of the R group  plays unique roles in enzymatic catalysis.
 only one amino group and one carboxyl group = NEUTRAL
 MONOAMINO-MONOCARBOXYLIC AMINO ACID AMINO ACIDS WITH POSITIVELY CHARGED POLAR R GROUPS
 Base (amino group) will neutralize the acid (carboxyl group) (BASIC AMINO ACIDS)
 All are neutral, EXCEPT:
BASIC ACIDIC
 Lysine  Aspartic Acid/
 Arginine Aspartate
 Histidine  Glutamic Acid/
Glutamate

 BASIC AMINO ACID LYSINE (Lys-K) ARGININE (Arg-R) HISTIDINE (His-H)

 if R-group has another amino group
 2 amino groups (base) versus 1 carboxyl group (acid) D. Amino Acids with NEGATIVELY CHARGED POLAR R GROUPS
=BASIC (ACIDIC)
 DIAMINO-MONOCARBOXYLIC AMINO ACID  Aspartic Acid
 Glutamic Acid
 ACIDIC AMINO ACID
 if R-group has another carboxyl group
 2 carboxyl groups (acid) VS 1 amino group (base) = ACIDIC
 MONOAMINO-DICARBOXYLIC AMINO ACID

3 TYPES OF POLAR AMINO ACIDS:

 Uncharged Polar
 Positively Charged Polar
 Negatively Charged Polar

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 PROTEIN CHEMISTRY | BIOCHEM A |Sources: Dra. Santos Lecture; LEA THERESE R. PACIS’ trans

All AAs have trivial or common names. Usually, derived from the  TYPES OF INTERACTIONS ENTERED INTO BY THE DIFFERENT
source from which they were first isolated: SIDE CHAINS OR R GROUPS OF AMINO ACIDS: “R to R”
 ASPARAGINE
 First amino acid to be discovered; 1. HYDROGEN BONDING
 Found in asparagus in 1806 a. –OH … O=C
 GLUTAMATE R-group with an –OH VS an R-group with a –COOH:
 First isolated from wheat gluten  Amino Acids with –OH in the R-group:
 TYROSINE  SERINE, THYROSINE, and THREONINE
 First isolated from “tyros”, means cheese  Amino Acids with COOH in the R-group:
 GLYCINE  ASPARTIC ACID and GLUTAMIC ACID
 From “glykos”, means sweet – for its taste Ex. Serine – Glutamic Acid

 ABBREVIATIONS OF AMINO ACIDS b. –OH…O

R-group with an –OH VS another R-group with an –OH
Ex. Serine – Serine, Serine – Threonine

c. –SH…O
R-group with an –SH VS an R-group with an –OH
 Amino Acids with –SH in the R-group:
 CYSTEINE
Ex. Cysteine – Serine

2. Ionic Interaction - -COO...NH3+

R-group with a -COOH VS an R-group with a NH3+
 Between an acidic (-COOH) amino acid and a basic (NH3+) AA
 Acidic AAs: ASPARTIC ACID and GLUTAMIC ACID
 Basic AAs: LYSINE, ARGININE, and HISTIDINE
Ex. Aspartic Acid – Arginine

3. Hydrophobic interaction – between nonpolar R groups

 Nonpolar Amino Acids: ALANINE, GLYCINE, VALINE,
LEUCINE, ISOLEUCINE, METHIONINE, PROLINE,
PHENYLALANINE, and TRYPTOPHAN

4. Disulfide bond
 Between sulfur and sulfur
Ex. Cysteine – Cysteine
KNOW HOW TO ABBREVIATE AMINO ACIDS
3-Letter-Abbreviation
- usually the first 3 letters of the amino acid STUDYING AMINO ACIDS
EXCEPT: 4 things to remember:
 Ile - Isoleucine 1. Know the structural formula
 Asn - Asparagine 2. Abbreviate (3-letter-abbreviation)
 Gln - Glutamine 3. How to classify
 Trp - Tryptophan  Is it Polar or Nonpolar?
 If it is POLAR, is it Charged or Uncharged?
 If it is CHARGED, is it Positive or Negative?
 SELENOCYSTEINE = 21st L--amino acid 4. Type of R-R Interaction
 a selenium (Se) atom replaces the
sulfur of its structural analog,
IDENTIFY THE TYPE OF INTERACTION
CYSTEINE.
1. Glutamic Acid and Serine
 a rare amino acid residue that is
 Glutamic Acid = Amino acid with –COOH in the R-group
inserted into polypeptide during
 Serine = Amino acid with –OH in the R-group
translation rather than created through
 Type of Interaction = HYDROGEN BONDING (-OH VS COOH)
postsynthetic modification
2. Aspartic Acid and Serine
 Unlike the other 20 genetically encoded
 Aspartic Acid = Amino acid with –COOH in the R-group
amino acids, it is not specified by a
 Serine = Amino acid with –OH in the R-group
simple 3-letter codon
 Type of Interaction = HYDROGEN BONDING (-OH VS COOH)
 present at the active site of several human enzymes that
3. Aspartic Acid and Lysine
catalyse Red-Ox reactions (Thioredoxin, Glutathione
 Aspartic Acid = Acidic Amino Acid (-COOH)
Peroxidase, Deiodinase)
 Lysine = Basic Amino Acid (NH3+)
 Impairment in human selenoproteins have been implicated
 Type of Interaction = IONIC BONDING (-COOH VS NH3+)
in TUMORIGENESIS and ATHEROSCLEROSIS, and are
4. Methionine and Alanine
associated with selenium deficiency cardiomyopathy
 Methionine = Nonpolar
(KESHAN DISEASE)
 Alanine = Nonpolar
 Fatal, congestive cardiomyopathy (Abnormality of the cardiac
 Type of Interaction = HYDROPHOBIC BONDING (Nonpolar
muscles due to selenium deficiency)
VS Nonpolar)
 Primarily affects children and women of childbearing age
 Occurs in areas with low soil trace elements (Parts of China,
New Zealand and Finland)  PHYSICAL PROPERTIES OF AMINO ACIDS

1. SOLUBILITY
 All amino acids are soluble in water, HOWEVER!

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 PROTEIN CHEMISTRY | BIOCHEM A |Sources: Dra. Santos Lecture; LEA THERESE R. PACIS’ trans

- R groups of: (most soluble to least)  amino and carboxylic acid groups of AAs readily ionize
1) polar, positively (basic amino acids) or negatively - pK values of the -carboxylic acid groups = lie in a small range
(acidic amino acids) charged are the most soluble/ around 2.2
hydrophilic - pK values of the -amino groups = all near 9.4
2) polar, uncharged amino acids  At physiological pH (7.4)
3) nonpolar amino acids with Short Rgroups - amino groups are protonated
4) nonpolar amino acids with long Rgroups - carboxylic acid groups are in their conjugate base
(carboxylate) form
POLARS (Most soluble in H2O) > NONPOLARS with SHORT R - Thus, AA can act as both an acid and a base.
GROUP > NONPOLARS with LONG RGROUP (Least soluble in H2O)
ARRANGE FROM MOST TO LEAST SOLUBLE IN WATER: Those -amino acids having a single amino group and a single
carboxyl group crystallize from neutral aqueous solutions as fully
ARGININE, ALANINE, AND ISOLEUCINE ionized species known as ZWITTERIONS (German for “hybrid ions”),
1. Arginine each having both a positive and a negative charge. These ions are
2. Alanine electrically neutral and remain stationary in an electrical field.
3. Isoleucine
ISOELECTRIC POINT (pHI or pI or IpH)
- pH at which an amino acid bears no net charge; hence, does not
move in an electrical field.
- pH exactly at the midpoint between the pK values on either side of
the zwitterion species.
- General rule regarding isoelectric points:
 IpH of neutral amino acids = neighborhood of 6.0
 IpH of acidic amino acids = very much below 6.0
 IpH of basic amino acids = very much above 6.0

LYSINE, GLYCINE, and METHIONINE  Physical properties of AAs - influenced by the ionic states of the
1. Lysine – Polar; positively charged -carboxyl and -amino groups and any ionisable groups in the
2. Glycine – Nonpolar; shorter R group side chains.
3. Methionine – Nonpolar; longer R group  Seven of the common AAs have ionisable side chains with
measurable pKa values.
2. MELTING POINTS  Each AA has either two or three pKa values = differ among AAs
Amino acids have a high melting point - usually 200C and above.  At a given pH, AAs have different net charges
 Ionic states of amino acid side chains strongly influence the 3D-
structures and biochemical functions of proteins.
3. TASTE
Taste could either be sweet, bitter, or tasteless.
 DIFFERENT FORMS OF AN AMINO ACID:

4. APPEARANCE 1. UNIONIZED FORM

They are white-crystalline in appearance.
 Carboxyl Group = NOT NEGATIVE (Didn’t
donate any proton)
5. OPTICAL PROPERTY
 Amino Group = NOT POSITIVE (Didn’t
- for all the standard amino acids, except Glycine, the -carbon
accept any proton)
is asymmetric, bonded to four different substituent groups:
 No charges
-carboxyl group, an amino group, an R group, and a hydrogen
 Unionized form does not exist in the body
atom.
– only used in writing the formula of the AAs
- -carbon is a chiral center
- Molecules with chiral center = OPTICALLY ACTIVE
i.e., they can rotate the plane-polarized light either to the right 2. DIPOLAR ION or ZWITTERION FORM
(dextrorotatory) or to the left (levorotatory).  Carboxyl Group = NEGATIVELY CHARGED
 Amino Group = POSITIVELY CHARGED
6. ULTRAVIOLET ABSORPTION SPECTRUM OF AROMATIC  Equal amounts of positive charge and
AMINO ACIDS negative charge = ELECTRICALLY
- AAs do not absorb visible light = thus, colorless, HOWEVER! NEUTRAL (ZERO CHARGE)
- Tyrosine, phenylalanine, and tryptophan absorb high  Dipolar Ions (ZWITTERION) form exist in
wavelength ultraviolet (UV) light the body
 Tryptophan  When you place it in an electrical field (cathode and anode), it
- absorbs UV light about 10x more efficiently than other 2; will not move = stay in the center because it is neutral in
- thus, makes the major contribution to the ability of most charge
proteins to absorb light in the region of 280 nm.
Aromatic Amino Acids (Tyr, Phe, Trp) 3. FULLY PROTONATED
- responsible for UV light absorption. - Fully protonated means all hydrogen ions are attached
- TRYPTOPHAN exhibits the most UV light absorption.  Carboxyl Group = INTACT; not negative
 Amino Group has another hydrogen
7. ACID-BASE PROPERTIES attached = POSITIVELY CHARGED (NH3+)
RECALL:  Fully protonated forms are ALWAYS
Amino Group = can accept a proton to become positively POSITIVE
charged  Form at pH 1 = ACIDIC MEDIUM
Carboxyl Group = can donate a proton to become negatively
charged

pK value - pH of the dissociation constant

- stronger acids dissociates faster than weak acids

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 PROTEIN CHEMISTRY | BIOCHEM A |Sources: Dra. Santos Lecture; LEA THERESE R. PACIS’ trans

4. FULLY DISSOCIATED FORM TITRATION OF ALANINE (NEUTRAL AMINO ACID)

- Fully dissociated means all hydrogen
ions are removed  At pH 1 = Ala is +1
 Carboxyl Group = NEGATIVELY - Titration starts with the most positive form.
CHARGED - adding Sodium Hydroxide (NaOH) to a medium that started
 Amino Group = NOT POSITIVE from pH 1 will result to a higher pH.
 Fully dissociated forms are ALWAYS  pH 7 = Neutral
NEGATIVE   pH 7 = Acidic
 Form at pH 11 and above = ALKALINIC MEDIUM   pH 7 = Basic
- NaOH has a negatively charged –OH that will attract a
 TITRATION OF AMINO ACIDS or Protonic Equilibria hydrogen ion.
Protonic equilibria
- the step by step dissociation of the amino acid, starting from the *It has 2 hydrogen ions that it can attract.
fully protonated/positive form up to the fully dissociated/negative Which one will it attract first?
form. - depends on the strength of the acids,
DISSOCIATION CONSTANT, the stronger
the acid, the higher the dissociation constant;
vice versa. Thus, if the acid is stronger, it will
dissociate faster.
 USES:
i. In this case, CARBOXYL GROUP (-COOH) will donate
1. it can predict the charge of the amino acid in a given
proton first. This dissociation is designated as pK1.
solution with known pH.
- The higher the dissociation constant, the lower the pK value.
2. it can devise a procedure of separating amino acids
(If pH = more acidic = dissociation constant = pK value)
based on their charges.
- pK1 = always represents the dissociation of the
3. From the titration curve, one can know the regions of
ALPHACARBOXYL GROUP (pH2.3 for Ala)
buffering power of the amino acid. These are the relatively
 Upon dissociation, (COOH COO-) = Ala is ZWITTERION
flat portions of the curve, extending for approximately 1 pH
ii. As more base is added, the H in the AMINO GROUP will now
unit on either side of the pK values.
dissociate, designated as pK2.
- pK2 = always represents the dissociation of the ALPHAAMINO
A. TITRATION OF NEUTRAL AMINO ACIDS
GROUP (pH9.7 for Ala)
Ex. ALANINE (Ala)
 Upon dissociation, (NH3+ NH2), = Ala is -1.
- During titration with a strong base (such as NaOH), Ala loses
two protons, in a stepwise fashion
- In a strongly acidic solution (pH 1), Ala is present mainly in the pK VALUES OF ALANINE:
form in which the carboxyl group is uncharged or  pH of pK1 = 2.3
undissociated, having a net charge of +1 (since ammonium - means that if the pH of the medium is at exactly 2.3, the
group is protonated) form of Ala is 50% +1 and 50% Zwitterion (half will be
- Lowering H+ concentration results in carboxyl group losing its fully protonated, half will be dissociated)
proton = to become a negatively charged carboxylate - above 2.3, predominant form of Ala is Zwitterion
group; (NOTE: Protons are first lost from the group with the - below 2.3, predominant form of Ala is +1
lowest pKa)  pH of pK2 = 9.7
- at ISOELECTRIC POINT, ala has no net charge and is - means that if the pH of the medium is at exactly 9.7, the
electrically neutral (AAs are least soluble at this pH) form of Ala is 50% Zwitterion and 50% -1.
- above 9.7, predominant form of Ala is -1
ISOELECTRIC POINT (IpH): - below 9.7 & above 2.3, predominant form of Ala is Zwitterion
- pH wherein an amino acid is 100% Zwitterion

 Rule regarding Isoelectric Points:

 Neutral Amino Acids = Have an IpH around 6 (6± = 5-7)
 Acidic Amino Acids = Have an IpH < 5 B
 Basic Amino Acids = Have an IpH > 7

Isoelectric point for alanine may be calculated as follows:

I

- IpH of Ala is 6.0; pH wherein Ala is 100% Zwitterion

- As the titration continues, ammonium group loses its TITRATION CURVE OF ALANINE
proton, leaving an uncharged amino group, ala then has a net - Note that pK values are not connected by a straight line
negative charge because of the carboxylate group. - but flattened around the pK values (A, B) – this indicates that
there is a buffering capacity of the AAs at the pK values
- AAs are good buffers at their pK values = buffering zones
- no buffer effect at the isoelectric point (I) (Straight/Steep)

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 PROTEIN CHEMISTRY | BIOCHEM A |Sources: Dra. Santos Lecture; LEA THERESE R. PACIS’ trans

EXERCISE (ALANINE):
 pK1 = 2.3
 pK2 = 9.7
 IpH = 6.0

1. What is the predominant form of Alanine if it is placed in a

medium with pH5?
= Ala is ZWITTERION; Because 5 is above 2.3 (pK1) and
below 9.7 (pK2) IpH of Glu is 3.2; pH wherein Glu is 100% Zwitterion

2. What are the exact values wherein Alanine is a good buffer?  Rules for IpH:
= 2.3 and 9.7;  Neutral Amino Acids = Have an IpH around 6 (6± = 5-7)
- Amino acids are good buffers at their pK values.  Acidic Amino Acids = Have an IpH < 5 - satistfied
 Basic Amino Acids = Have an IpH > 7
B. Titration of ACIDIC AMINO ACID
Ex. GLUTAMIC ACID (Glu)
EXERCISE (GLUTAMIC ACID):
 pK1 = 2.2
 pKr= 4.3
 pK2 = 9.6
 IpH = 3.2

1. What is the predominant form of Glu if it is placed in a

medium with pH4?
= Glu is zwitterion; Because 4 is above 2.2 (pK1) and
below 4.3 (pKr)
AAs with ionizable side chains have more complex titration curves.
Ex. 2. What are the exact values wherein Glu is a good buffer?
 Glutamic acid has a carboxyl side chain/ Rgroup. = 2.2, 4.3 and 9.6
- At low pH, Glu has a net charge of +1. - Amino acids are good buffers at their pK values.
- As base is added, -carboxyl group loses a proton to
become a carboxylate group (COO-)
- Glu now has no net charge.
- As still more base is added, second carboxyl group loses a
proton, and the molecule has now a negative (-1) charge.
- Adding additional base results in the ammonium ion losing
its proton.
- At this point, glutamate has a net charge of –2.
- The IpH for glu is the pH halfway between the pK values for the
two carboxyl groups. (Source: Biochemistry by McKee & McKee;
2nd ed. Chapter 5 – peptides and proteins)
TITRATION OF GLUTAMIC ACID (ACIDIC AMINO ACID)

- In Glu, there is another carboxyl group at the Rgroup, thus, 3

groups to dissociate.

 At pH 1 = Glu is +1
- Fully Protonated Form – most positive form of glu HOW TO USE THE pK VALUE TABLE*
 pK1 = Always for the ALPHA-CARBOXYL GROUP
i. At pK1, -CARBOXYL GROUP will dissociate first. (pH2.2 for  pK2 = Always for the ALPHA-AMINO GROUP
Glu)  pKr = Always for the R-GROUP
 Results to (-COOH  COO-), = Glu is ZWITTERION
- Lowest pK value = will dissociate first
- As more base is added, either Rgroup or the amino group
that will get dissociated next.  In BASIC AMINO ACIDS, pK of -Amino Group is < or 
- In this case, the CARBOXYL GROUP from the RGROUP will than pK of the R-Group thus, -Amino Group dissociates first.
now get dissociated (NOTE: Acids dissociate first.) (for lysine and arginine)
pK1 (a-COOH) pK2 (a-NH2) pKr (R-Group)
ii. At pKr, carboxyl group of the Rgroup gets dissociated. (pH4.3 Histidine 1.8 9.3 6.0
Lysine 2.2 9.2 10.8
for Glu)
Arginine 1.8 9.0 12.5
 Results to (-COOH  COO-), Glu is -1. *Values from Harper’s Illustrated Biochemistry 30th Edition Table 3-1
Note: in Basic AAs, fully protonated form is +2, due to the
iii. At pK2, -AMINO GROUP will disassociate last. (pH9.6 for other amino group in the R-group.
Glu)  In ACIDIC AMINO ACIDS, pK of R-Group is < than pK of
 Results to (NH3+  NH2), Glu is -2. -Amino Acids = R-Group dissociates first.
 In all the amino acids, the -CARBOXYL GROUP has the
NOTE: In all the amino acids, ONLY ACIDIC AMINO ACIDS lowest pK value; thus, it always dissociates first.
CAN BECOME -2, due to their 2 carboxyl groups.
*DON’T MEMORIZE, pK values will be provided and may slightly
In computing for IpH of Glu, pK values will be added before and differ from this source. Analyze numbers carefully.
after the zwitterion ion. Thus, pK1 and pKr.
REMEMBER: LOWEST pK values will dissociate first.

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 PROTEIN CHEMISTRY | BIOCHEM A |Sources: Dra. Santos Lecture; LEA THERESE R. PACIS’ trans

III.PEPTIDES Vasopressin (CYS-TYR-PHE-GLN-ASN-CYS-PRO-ARG-GLY-NH2)

- known as antidiuretic hormone (ADH)
Peptides - secreted in response to low pressure or a high Na+ concentration.
- Has a low molecular weight; - stimulates the kidneys to retain water = concentrated urine
- Typically consists less than 50 amino acids
3. MET-ENKEPHALIN & LEU-ENKEPHALIN
Protein - relieves pain
- Molecules with more than 50 amino acids - belong to a group of peptides called OPIOID PEPTIDES
- Consists one or more polypeptide chains - Opioid peptides
 predominantly found in nervous tissue cells
Oligopeptides  molecules that relieve pain and produce pleasant sensations
- Polymers consisting of 2 to 10 amino acids  bind to receptors in certain cells of the brain and induce
 Dipeptide = 2 amino acids analgesia (deadening of pain sensations).
 Tripeptide = 3 amino acids  represent one of the body’s own mechanisms for the control of
pain.
Polypeptides - Pentapeptides that differ only in their C-terminal amino acid
- Amino acid polymers with molecular weights ranging from several residues.
thousand to several million daltons.  Met-enkephalin = TYR-GLY-GLY-PHE-MET
- Have more than 10 amino acid residues (More than 10, Below 50)  Leu-enkephalin = TYR-GLY-GLY-PHE-LEU
- Peptides perform prominent roles in the neuroendocrine system as - Enkephalin receptors also bind morphine, heroin and other
hormones, hormone-releasing factors, neuromodulators or addicting opiate drugs (although these are not peptides).
neurotransmitters.
4. ATRIAL NATRIURETIC FACTOR
 Peptides with significant biological activities - peptide with 28 amino acid residues
- produced by specialized cells in the heart and nervous system
- stimulates the production of a dilute urine (effect opposite to
1. GLUTATHIONE - -glutamyl-L-cysteinylglycine
vasopressin)
- Synthesized by humans in their body
- Made up of Glutamic Acid, Cysteine, Glycine 5. SUBSTANCE P & BRADYKININ
- most important function: detoxifying effect - stimulate the perception of pain (effect opposed by opioid
 detoxifies Hydrogen Peroxide (H2O2) (normally produced in peptides)
RBCs, toxic when accumulated.) - Pain is a protective mechanism in animals that warns of tissue
 Glutathione converts H2O2 to water, controlling its toxicity. damage.
 Lacking/No Glutathione  accumulated H2O2  destroys
RBC membrane  hemolysis  hemolytic anemia 6. GLUCAGON
- antioxidant property - pancreatic hormone
 Glutathione must be reduced for it to function as an - has 29 amino acid residues
antioxidant. - increases blood sugar
 Whitening effect = side effect - opposite to the effect of insulin.
- Contains an unusual ϒ-amide bond
 wherein the ϒ-carboxyl group of the glutamic acid 7. CORTICOTROPIN
residue, not the -carboxyl group, contributes to the - contains 39 amino acid residues
peptide bond - increases blood sugar
- Found in almost all organisms - hormone of the anterior pituitary gland that stimulates the
- involved in many important biological processes: adrenal cortex
 protein and DNA synthesis; drug and environmental toxin
metabolism; and amino acid transport 8. L-ASPARTYLPHENYLALANYL METHYL ESTER
 Effective reducing agent; (reducing component is –SH group - commercially produced dipeptide: Phenylalanine+Aspartic Acid
of the cysteine residue; reduced glutathione is GSH) - an artificial sweetener better known as ASPARTAME or
- protects cells from the destructive effects of oxidation by NUTRASWEET.
reacting with substances such as peroxides (R-O-O-R, by
products of O2 metabolism)  STRUCTURE OF PROTEINS
Ex. In RBCs, hydrogen peroxide (H2O2) oxidizes the iron of
hemoglobin to its ferric form (Fe+3)  produces I. FORMATION of the peptide bond
Methemoglobin (incapable of binding oxygen) - the most important biochemical reaction of amino acids
= Glutathione - protects against the formation of
methemoglobin by reducing H2O2 in a reaction catalyzed Ex. Formation of dipeptide Alanyl-Serine:
by the enzyme glutathione peroxidase.

2. OXYTOCIN & VASOPRESSIN

- amino acid sequences of the 2 differ only by two residues.
- Contains 9 amino acids
- produced by the cleavage of polypeptide precursors within
different specialized cells in the hypothalamus
- After synthesis, they are transported down the nerve tracts into
the Posterior Pituitary Gland (where they are stored)
- secreted in response to specific signals from the hypothalamus
i. React 1 carboxyl and 1 amino group.
Oxytocin (CYS-TYR-ILE-GLN-ASN-CYS-PRO-LEU-GLY-NH2) ii. Remove water - DEHYDRATION REACTION
- stimulates: - -OH comes from your COOH
 contraction of uterine muscles during childbirth - H+ come from your NH2
 ejection of milk by mammary glands during lactation
- in males, oxytocin may have a regulatory role in the synthesis of
the sex hormone testosterone.

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 PROTEIN CHEMISTRY | BIOCHEM A |Sources: Dra. Santos Lecture; LEA THERESE R. PACIS’ trans

2. Relatively fewer titratable amino and carboxyl

functions
- less two titratable groups (1 amino and 1 carboxyl) for every
peptide bond formed.
ALANYL-SERYL-LYSYL-ASPARTYL-METHIONINE:

 Amino acids can be polymerized to form chains;

 represented as CONDENSATION REACTION - elimination of a water
molecule.
 Results in a CO-NH linkage, an amide linkage, is known as the
peptide bond.
 Amino Acid Residues - individual amino acids (the monomeric units)
incorporated into a peptide
SEPARATELY: TOTAL =12 titratable groups
Alanine, Serine, & Methionine – 2 titratable groups each
(Carboxyl Group and Amino Group) = 6 groups
Lysine - 3 titratable groups (Carboxyl Group, Amino Group,
and Amino Group at the R-Group)
Aspartic Acid - 3 titratable groups (Carboxyl Group, Amino
Group, and Carboxyl Group at the R-Group)

With peptide bonds: Total = 4 Titratable Groups

 12 Total Titratable Groups
(when amino acids are taken separately)
 Variations in the length and the amino acid sequence of polypeptides  4 Peptide Bonds X 2 = 8
contribute to the diversity of the shape and biological functions of (2 titratable grps used = 1 Carboxyl Group + 1 Amino Group)
proteins. = 12-8 =4
 Proteins are molecules that contain one or more polypeptide chains.
3. Hydrolyzed by enzymes which are specific for the
Polypeptides are linear polymers. peptide bond
- Each amino acid participates in two peptide bonds and is linked Ex. pepsin, trypsin, chymotrypsin, etc.
to its neighbors in a head-to-tail fashion rather than forming Enzymes are specific when it comes to the type of bond
branched chains that they react on.
- Residues at the two ends of the polypeptide each participate in
just one peptide bond
4. X-ray diffraction studies proved the existence of peptide
 AMINO TERMINUS/ N-TERMINAL - residue with a free
linkages between amino acids in hemoglobin and myoglobin
amino group (leftmost residue)
 CARBOXYL TERMINUS/ C-TERMINAL - residue with a free
5. Synthesis of insulin - by combining AAs to the peptide bond
carboxylate group (rightmost)
 LEVELS OF STRUCTURAL ORGANIZATION OF PROTEINS
II. NOMENCLATURE
- how peptide chains are named:
A. PRIMARY STRUCTURE (by peptide bonds)
 By convention, peptides are named based on the sequence of
 Quantitative amino acid composition
their constituent amino acids:
- beginning at the left (N-terminal residue) towards the right (C- State how many AAs are there.
terminal residue).  Sequence of amino acids – from first to last
 Amino acid residues in polypeptides are named by: State all AAs from the N-terminal down to C-terminal
i. dropping suffix -ine or -ate of all AA except C-terminal*  Number of peptide chains
ii. replacing it by –yl
*C-terminal residue/ Last amino acid - retains its original name. Ex.
Ex. ALA-SER-LYS (Alanine-Serine-Lysine) 1. Describe the Primary Structure of the chain below
= Alanyl-Seryl-Lysine Ila-Leu-Asp-Arg-Trp-Lys-Ser-Cys-Val-Glu-Ile-Gln-
His-Met-Ala-Pro-Tyr-Asn-Gly-Thr
-INE ending - There are 20 Amino Acids
- indicates that its -carboxyl group is not involved in peptide - Isoleucine on the N-terminal followed by Leucin, Aspatic Acid,
bond formation Arginine and so on. Threonine on the C-Terminal
- 1 Peptide Chain
Ex. SER-ALA-LYS-PHE-GLU-TYR
Seryl-Alanyl-Lysyl-Phenylalanyl-Glutamyl-Tyrosine 2. Describe the Primary Structure of the Chain Below

- 20 amino acids divided into 2 peptide chains

III. EVIDENCES that a PEPTIDE LINKAGE EXIST in proteins - First peptide chain has 10 amino acids.
- Arginine on the N-Terminal followed by Aspartic Acid,
1. Positive reaction to the Biuret test Phenylalanine, Leucine, Glycine, Tryptophan, Cysteine, Alanine,
 Biuret Test = general test for proteins Methionine, Glycine at the C-Terminal
- Becomes positive in at least 2 peptide bonds - Second Peptide chain has 10 amino acids.

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 PROTEIN CHEMISTRY | BIOCHEM A |Sources: Dra. Santos Lecture; LEA THERESE R. PACIS’ trans

- Glutamine at the N-Terminal followed by Glycine, Serine,  Important features of the alpha-helix:
Valine, Threonine, Cysteine, Glutamic Acid, Histidine, Alanine, 1. Stabilized by inter-residue hydrogen bonds formed between
Phenylalanine at the C-Terminal the H atom attached to a peptide N and the carbonyl O of the
- 2 peptide chains linked by a Disulfide bridge fourth residue down the polypeptide chain. (intrachain)

 In general, proteins contain at least 40 amino acid residues

(polypeptides smaller than that are simply called peptides)
 40 residues - minimum for a polypeptide chain to fold into a
discrete and stable shape that allows it to carry out a particular
function.
 Each AA has characteristic chemical and physical properties, thus its
presence at a particular position influences the properties of that
protein.
 backbone or main chain of a protein – alternating -carbon and
peptide bond; atoms that participate in peptide bonds; ignoring the
side chains of the amino acid residues.

Sequence of amino acid residues

- most important element of primary structure
- function of a protein depends upon its amino acid sequence
- Each type of protein has a unique amino acid sequence.
- plays a fundamental role in determining the 3-dimensional
structure of the protein and its function.
- Proteins with different functions always have different amino acid
sequences. 2. Each peptide bond participates in the hydrogen bonding. This
 Thousands of human genetic diseases have been traced to the confers maximum stability.
production of defective proteins. The defect can range from a 3. An alpha-helix forms spontaneously as it is the lowest
single change in the amino acid sequence (as in sickle cell energy, most stable conformation for a polypeptide chain. The
anemia), to deletion of a larger portion of the polypeptide chain alpha-helix is relatively stable, provided the R groups of the
(as in most cases of Duchenne muscular dystrophy). polypeptide chain are uncharged and incapable of hydrogen
- Therefore, if the primary structure is altered, the function of bonding.
the protein may also be changed. 4. There are about 3.6 amino acid residues per turn and the
distance between corresponding points per turn (pitch) is .54 nm
B. SECONDARY STRUCTURE or 5.4 A. In this coil, the spacing per residue is about 1.5 A or
- due to the formation of hydrogen bonds between peptide .15 nm. (5-2)
bonds
- two types:
a. coils or helices
- brought about by INTRACHAIN hydrogen bonding
Intrachain Hydrogen Bonding
- hydrogen bond between peptide bonds that are not
adjacent, as much as possible it will be atleast 4 amino
acids away.
- Peptide chain must bend to reach another peptide bond,
resulting to the formation of coils/helices.

Two types of coils:

a) Alpha-helix – regularly coiled; same intervals – 3.6
AA per turn, 5.4 A distance; most stable form of the
secondary structure; maximally hydrogen bonded.
b) Random Coil – irregularly coiled, intervals are not the
same; very unstable.
5. Amino acid R groups extend outward from the helix. (5-3)
b. sheets or pleats
- brought about by INTERCHAIN hydrogen bonding
As much as possible, a protein would like to assume the alpha-helix
Interchain Hydrogen Bonding form; however, there are factors that prevents this formation.
- between different chains;
- hydrogen bonds between peptides bonds belonging to
 Factors that destabilize the Alpha-helix:
different chains;
a. Presence of adjacent similarly charged amino acids
- resulting to chains closer to one another
Similarly charged:
 Similarly acidic – Aspartic Acid(Asp), Glutamic Acid (Glu)
Two types of sheets:
 Similarly basic – Lysine (Lys), Arginine (Arg), Histidine (His)
a) Parallel pleats – same direction for all the chains, ex.
“all N- terminals on top, all C- terminals at the bottom” If you find these AAs adjacent in a chain, this will not make
b) Anti-parallel pleats – directions are not the same, the alpha-helix.
ex. “one chain N-terminal is on top, the next has the C-
terminal on top.”; more stable than parallel b. Presence of adjacent bulky R groups of amino acids
Branched-chain amino acids
ALPHA-HELIX - have bulky Rgroups
- discovered by Linus Pauling in 1951; ranks as one of the - Valine (Val), Leucine (Leu), Isoleucine (Ile)
landmarks of structural Biochemistry
- MOST STABLE form of the secondary structure, because it is c. Presence of Proline
regularly coiled.

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 PROTEIN CHEMISTRY | BIOCHEM A |Sources: Dra. Santos Lecture; LEA THERESE R. PACIS’ trans

PROLINE  Factors that destabilize the beta-pleated sheet:

Whenever found inside a peptide chain, it breaks the helix. 1. Bulky R groups
- the least common residue in an alpha-helix 2. R groups with like charges
- “helix breaker”
 its rigid, cyclic side chain disrupts or bends the right-handed C. TERTIARY STRUCTURE OF PROTEINS (R-R groups)
helical conformation by occupying space that a neighboring - 3D structure; protein conformation; or shape of protein
residue of the helix would otherwise occupy - results from the further folding of the secondary structure
 contains a rigid ring that prevents the N-C bond from rotating - maintained by R-R interactions
 lack of a hydrogen atom on its amide nitrogen keeps proline - overall snake-like appearance (alpha-helix/random
from fully participating in the hydrogen-bond network of an intervals)
alpha-helix = no intrachain H bonds - refers to the unique 3-dimensional structure of the protein,
- found more often at the ends of alpha-helices than in the interior including the geometric relationship between distant segments of
due to the factors mentioned. the primary structure and the relationship of the side chain
 It disrupts the conformation of the helix, producing a bend, group with respect to each other in 3-dimensional space.
when present elsewhere - results from the folding of a polypeptide which may already
possess some regions of alpha-helix and/or beta-sheet, into a
Exercise: Will it be alpha-helical all through out? closely packed, nearly spherical shape.
(Always start with the N-terminal) In this structure, AAs that are far from each other, will
become closer to one another because of its globular shape.
VAL-LYS-ASP-TRP-SER-THR-MET-ALA-PRO-GLU-LYS-TYR- - it indicates how secondary structural features - helices, sheets,
ASN-ASP-GLU-HIS-LEU-ILE-MET-CYS-LYS-ARG-ALA-ALA bends, turns, and loops assemble to form domains and how
these domains relate spatially to one another.
- VAL-LYS-ASP-TRP-SER-THR-MET-ALA-PRO-GLU-LYS-TYR-
ASN-ASP-GLU-HIS-LEU-ILE-MET-CYS-LYS-ARG-ALA-ALA  Two general classes of proteins based on tertiary
 PRO – proline structure:
 ASP-GLU – adjacent similarly acidic a) Fibrous proteins
 LEU-ILE – adjacent bulky Rgroups - serve mainly structural roles, have simple repeating
 LYS-ARG – adjacent similarly basic elements of secondary structure.
b) Globular proteins
BETA-PLEATED SHEET: - have more complicated tertiary structures, often containing
- second type of commonly occurring protein secondary structure. several types of secondary structure in the same
- Sheets are formed when two or more almost fully extended polypeptide chain.
polypeptide chains are brought together side by side so that regular * The first globular protein structure to be determined by
hydrogen bonds can form between the peptide backbone amide NH x-ray diffraction methods, was that of myoglobin.
and carbonyl oxygen groups of adjacent chains.
- Like the alpha-helix, β-sheets derive much of their stability from Domains or Lobes
hydrogen bonds between the carbonyl oxygen and amide hydrogen - group of AAs within the chain that performs one function.
of peptide bonds. Ex. In immunoglobulins, one of the domains is where the antigen
- Each individual segment is referred to as a β-strand. Rather than will attach
being coiled, each β-strand is fully extended. - discrete, independent folding units within the tertiary structure
- sufficient to perform a particular chemical or physical task such as
binding of a substrate or other ligand
- consist of combinations of several units of supersecondary structures
- motifs are usually adjacent in the primary structure
- size varies greatly from about 25 – 30 to about 300 amino acid
residues, with an average of about 100 amino acid residues.
- may have binding sites for one to three ligands.
- other domains may anchor a protein to a membrane or interact with
a regulatory molecule that modulates its function.

 Types of interactions that stabilize tertiary structure:

1. Hydrophobic interactions
- As a polypeptide folds, hydrophobic R groups are brought into
close proximity because they are excluded from water.

2. Electrostatic interactions or ionic interactions

- The strongest electrostatic interaction in proteins occurs between
ionic groups of opposite charge or “salt bridges”
 B-sheets can occur in two different arrangements:
1. antiparallel beta-sheet 3. Hydrogen bonds
- neighboring hydrogen bonded polypeptide chains run in - A significant number of hydrogen bonds form within a protein’s
opposite directions. interior and on its surface.
- more stable because fully colinear hydrogen bonds form. - In addition to forming hydrogen bonds with one another, the
2. parallel beta-sheet polar amino acid side chains may interact with water or with the
- hydrogen bonded chains extend in the same direction. polypeptide backbone.

 Features of the beta-pleated sheet: 4. Covalent bonds

1. Hydrogen bonds are interchain. - Covalent linkages are created by chemical reactions that alter
2. R groups lie above or below the zigzagging planes of the pleated a polypeptide’s structure during or after its synthesis.
sheet and are nearly perpendicular to them.  These reactions are sometimes referred to as post-
3. Amino acids with less bulky R groups are present. translational modifications.
- disulfide bridges - most prominent covalent bonds in tertiary
structure; found in many extracellular proteins.

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 PROTEIN CHEMISTRY | BIOCHEM A |Sources: Dra. Santos Lecture; LEA THERESE R. PACIS’ trans

PROTEIN FOLDING  PROTEIN ANALYSIS

- a cooperative, sequential process, in which formation of the first few - amino acid sequence in a polypeptide can be determined by
structural elements assists in the alignment of subsequent structural a process of partial breakdown into manageable
features. fragments, followed by stepwise analysis proceeding from
- folding pattern and final conformation of a protein - dependent one end of the chain to the other.
upon its primary structure. - Before proceeding with the different steps involved in
- Two types of proteins are required for proper folding of proteins protein analysis, see to it first that the protein you are
in cells: analyzing is purified.
1. Enzymes - Fortunately, variations in polypeptide size and chemical
2. Molecular Chaperones composition make it easier to devise methods to separate
- proteins that have the net effect of increasing the rate of proteins from each other and from other biological
correct folding by binding newly synthesized polypeptides molecules.
before they are completely folded.
- prevent the formation of incorrectly folded intermediates that  PROTEIN PURIFICATION precedes protein analysis
may trap the polypeptide in an aberrant form. - Purification is a mandatory step in studying macromolecules.
- can also bind to unassembled protein subunits, preventing - Proteins are purified by FRACTIONATION PROCEDURES.
them from aggregating incorrectly and precipitating before - In a series of independent steps, the various physicochemical
they are assembled into a complete multisubunit protein. properties of the protein of interest are used to separate
- appear to inhibit incorrect folding and assembly pathways by it progressively from other substances.
interacting with surfaces on the newly synthesized polypeptide - This idea is not necessarily to minimize the loss of the desired
chain that are exposed only during folding and assembly. protein, but to eliminate selectively the other components
- form stable complexes with the polypeptide chain, often of the mixture so that only the required substance
through interaction with hydrophobic regions. remains.
- assists in the translocation of polypeptide chains across
membranes and the assembly and disassembly of large Characteristics of proteins and other biomolecules that are
multiprotein structures. used in the various separation procedures:
- involved in escorting some proteins to specific locations within
the cell – “chaperone” Characteristic Procedures
- can also “rescue” proteins that have become Solubility - Paper chromatography
thermodynamically trapped in a misfolded dead end by - Thin layer chromatography
unfolding hydrophobic regions and providing a second chance Charge - Ion exchange chromatography
- Electrophoresis
to fold productively.
Polarity - Hydrophobic interaction chromatography
Alzheimer’s disease Size - Gel filtration chromatography
- SDS-PAGE electrophoresis
- caused by protein misfolding
- Ultracentrifugation
- from β-amyloid misfolding leading to formation of tangles and Binding specificity - Affinity chromatography
plaques

D. QUATERNARY STRUCTURE (between peptide chains)  PROTEIN SEQUENCING

- exhibited only by oligomeric proteins (containing more than Bovine Hormone Insulin
one peptide chain) - first protein whose sequence was determined
How one chain interacts with another chain. Thus, this structure - complete sequence was reported in 1953
needs atleast 2 peptide chains. - by Frederick Sanger (who later devised the chain-terminator
method of DNA sequencing)
Most proteins, particularly those with molecular masses above The amino acid sequences of thousands of polypeptides are now
100 kD, consist of more than one polypeptide chain. known.
 Subunit - each chain in an oligomeric protein;
- each polypeptide component;  Importance of Protein Sequencing:
- maybe identical or quite different. 1. Knowledge of a protein’s amino acid sequence is prerequisite
 Multimer - multisubunit protein for determining its 3-dimensional structure and is
 Oligomer - multimer with just a few subunits essential for understanding its molecular mechanism of
 Protomers - their identical units action.
 Dimers (2) and tetramers (4) - a large number of oligomeric 2. Sequence comparisons among analogous proteins from
proteins containing 2 or 4 subunit protomers different species yield insights into protein function and
 Monomeric proteins - consist of a single polypeptide chain. reveal evolutionary relationships among the proteins and
 Dimeric proteins - contain two polypeptide chains. the organisms that produce them.
3. Many inherited diseases are caused by mutations leading to an
- These chains or subunits are held together by noncovalent amino acid change in a protein. Amino acid sequence analysis
interactions such as: can assist in the development of diagnostic tests and
 hydrophobic interactions effective therapies.
- principal force holding the subunits together
 electrostatic interactions - Briefly, the protein must be broken down into fragments small
- contribute to the proper alignment of the subunits enough to be individually sequenced. The primary structure of the
 hydrogen bonds intact protein is then reconstructed from the sequences of
 interpolypeptide disulfide bonds overlapping fragments.
NOTE:
In tertiary structure, interactions are R to R or side chain to  STEPS INVOLVED IN PROTEIN ANALYSIS
side chain.
In quarternary structure, it is between peptide chains. I. Determine amino acid composition of the protein
- this structure designates the characteristic manner in which the a. breakdown the polypeptide chain into its constituent amino
individual, folded polypeptide chains fit each other in the native acids
conformation of an oligomeric protein. b. separate the resulting amino acids according to type
c. measure the quantities of each amino acid

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 PROTEIN CHEMISTRY | BIOCHEM A |Sources: Dra. Santos Lecture; LEA THERESE R. PACIS’ trans

f) THERMOLYSIN - heat-stable bacterial protease; cleaves off

A. Breakdown of the polypeptide chain into its component amino peptide bonds whose amino function is contributed by Leu,
acids can be done in 3 ways: Ile, Val
1. Acid hydrolysis Cuts peptide bonds where amino side is a branched-chain
2. Basic hydrolysis AA.
3. Enzymatic hydrolysis g) PAPAIN - found in certain plant tissues like papaya; cleaves
off peptide bonds whose carbonyl function is contributed by
1. Hydrolysis of Proteins by Acid Lys, Leu, Arg, Gly
- Heat proteins at 100 – 120C for 10 to 24 hours in 6N HCl h) BROMELAIN - derived from pineapple; cleaves off peptide
- Disadvantages of Acid hydrolysis: bonds whose carbonyl function is contributed by Lys, Ala,
 All the tryptophan and variable amounts of serine Tyr, Gly
and threonine are destroyed.
 Glutamine and asparagine are deamidated to Chemical reagents that promote peptide bond cleavage at
glutamate and aspartate specific residues
Deamidation - means converting the amide group a. Cyanogen bromide (CNBr) – cleaves peptide bonds whose
to a carboxyl group carbonyl part is contributed by Methionine; there will be
 Glutamic acid undergoes intramolecular dehydration to conversion of free-carboxyl terminal methionine to homoserine
form pyrollidone-5-carboxylic acid lactone
 Other amino acids may undergo intermolecular
dehydration forming cyclic anhydrides or b. Staphylococcus aureus V8 protease – cleaves peptide bonds
diketopiperazines whose carbonyl part is contributed by Glutamic acid, particularly
where the amino part is contributed by a hydrophobic amino acid
2. Hydrolysis of Proteins by Base [- - Glu – X (hydrophobic)]
- Incubate proteins with 2 to 4 N NaOH at 100C for 4 to 8 hrs
II. Determine N and C terminals
- Disadvantages of basic hydrolysis: Each polypeptide chain has an N-terminal and a C-terminal
 Cysteine, cystine, Ser, Thr and Arg are decomposed residue.
in the process. Identifying these end amino acids can establish the number of
 Other amino acids may be partially destroyed by chemically distinct polypeptides in a protein.
deamination.
Deamination - removing amino group; remaining Methods of determining the N-terminal amino acid in a
part is now called an -Keto acid. polypeptide chain
 Aspartic Acid – oxaloacetate
 Glutamic Acid – alpha-ketoglutarate 1. Sanger’s reaction or reaction with FDNB
 Alanine – pyruvate i. reacted with Sanger’s reagent or 1-Fluoro-2,4-
 Glycine – Glyoxylic acid dinitrobenzene (FDNB)
 Racemization of amino acids occur. ii. it will attach to N-terminal, converting peptide chain to a
DNP peptide.
To recover as much as possible all amino acids: iii. upon hydrolysis, all bonds will be cut
- It is best to divide your sample protein into 3 parts to iv. all AA will be separated, with the N-terminal still attached
perform acid, basic and enzymatic hydrolysis, respectively. to DNP (determining the N-terminal)
- In this way, whatever AA is destroyed from other - Frederick Sanger received the Nobel Prize in 1958 for being
hydrolysis can be recovered by another hydrolysis. the 1st one to determine the sequence of a polypeptide
Ex. In alkaline/basic hydrolysis, goal is TO RECOVER (bovine insulin).
TRYPTOPHAN, as it is destroyed completely in acid
hydrolysis.

3. Enzymatic Hydrolysis of Proteins

Using enzymatic hydrolysis alone is effective to determine
all AAs, however, very costly. Because of enzymes’
specificity as to what peptide bond they can only cut, 2. Dansyl chloride reaction
several enzymes will be needed to be able to cut all - Reaction with 1-dimethylaminonaphthalene 5-sulfonyl chloride
peptide bonds, thus very expensive. - same principle as Sanger’s reaction;
a) PEPSIN - cleaves off peptide bonds whose carbonyl - only the N-terminal amino acid is attached to the Dansyl
function is contributed by Phe, Tyr, Trp, Leu, Glu, Gln chloride and the rest of the amino acids are recovered as free
b) TRYPSIN - cleaves off peptide bonds whose carbonyl amino acids.
function is contributed by Lys and Arg
c) CHYMOTRYPSIN - cleaves off peptide bonds whose
carbonyl function is contributed by Phe, Trp, Tyr (Aromatic)
Pepsin, Trypsin, Chymotrypsin
- digestive enzymes used to digest proteins.
3. EDMAN’S REACTION
- exists in human’s body in their zymogen(inactive) form
- Pehr Edman introduced phenylisothiocyanate (Edman’s
 Pepsinogen - activated to Pepsin by acidity of stomach,
reagent) to selectively label the amino-terminal residue of a
HCl
peptide.
 Trypsinogen - activated to Trypsin by enterokinase
- In contrast to Sanger’s reagent, the phenylthiohydantoin (PTH)
 Chymotrypsinogen - activate to Chymotrypsin by trypsin
derivative can be removed under mild conditions to generate a
- only activated if there is a need
new amino terminal residue.
d) CARBOXYPEPTIDASE - cleaves off C-terminal amino acids - Successive rounds of derivatization with Edman’s reagent can
Cuts the last or C-terminal AA from the rest of the chain. be used to sequence many residues of a single sample
e) AMINOPEPTIDASE - cleaves off N-terminal amino acids of peptide.
Cuts the first or N-terminal AA from the rest of the chain.

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 PROTEIN CHEMISTRY | BIOCHEM A |Sources: Dra. Santos Lecture; LEA THERESE R. PACIS’ trans

- usually, the first 20 to 30 residues of a peptide can readily be

determined by the Edman’s method;  SEQUENATOR
- for large polypeptides, they must first be cleaved into smaller - a programmed machine performing Edman degradation.
peptides prior to Edman’s sequencing i. mixes reagents in the proper proportions
- Edman sequencing has been automated, using a thin film or ii. separates the products
solid matrix to immobilize the peptide and HPLC to identify iii. identifies them and records the results.
PTH amino acids.
IV. CHECK NUMBER OF POLYPEPTIDE CHAINS
Ex. of Edman’s reaction: - If there are two or more peptide chains, separate them.

A. Disulfide bonds
B. Noncovalent bonds - denaturing agents

Cleavage of disulfide bridges

1. Oxidation using performic acid
- leads to the formation of cysteic acids
2. Reduction using mercaptoethanol, thioglycollate and
sodium borohydride
- the disulfides are reduced to sulfhydryl form

V. CHROMATOGRAPHY / IDENTIFICATION

MASS SPECTROMETRY (MS)

- principal method for determining the sequences of peptides and
proteins due to its superior sensitivity, speed and versatility
- also used to determine the post-translational modification of proteins
by the addition or deletion of carbohydrate moieties, phosphoryl,
i. reacted with phenylisothiocyanate (Edman’s reagent) hydroxyl, or other groups
ii. attaches to N-terminal, converting peptide to PTC peptide. - discriminates molecules based solely on their mass, thus, it can
iii. Upon hydrolysis, it will only cut the first bond; separating the detect the comparatively subtle physical changes in proteins that
N-terminal, which becomes phenylthiohydantoin AA + rest occur during the life cycle of a cell or organism.
of the chain intact.
iv. Subjecting it to another Edman’s reagent will determine the TANDEM MASS SPECTROMETRY
second amino acid of the peptide chain; and so on. - employs the equivalent of two mass spectrometers linked in series
- can be used to screen blood samples from newborns for the
presence and concentrations of amino acids, fatty acids and other
4. Use of Leucine Aminopeptidase
metabolites. Abnormalities in metabolite levels can serve as
- Splits only the N-terminal amino acid from the polypeptide
diagnostic indicators for a variety of genetic disorders, such as
chain.
Phenylketonuria (PKU).
Methods of determining the C-terminal amino acid in a
METHODS OF SEPARATING AND IDENTIFYING AMINO ACIDS
peptide chain
I. PAPER CHROMATOGRAPHY
1. Reduction with Lithium Borohydride
- separation on the basis of differences in solubility of amino
- the C-terminal amino acid is converted to a primary alcohol
acids between two immiscible solvents.
Cuts all the AA separately, with the C-terminal converted to - the solvents usually used are:
an alcohol.  a hydrophobic organic solvent (mobile phase)
 water molecules bound to the cellulose (stationary phase)
2. Hydrazinolysis
- reaction with anhydrous hydrazine at 100C II. Thin layer chromatography (TLC)
- same principle as paper chromatography, but makes use of
glass plates for support.

III. Ion Exchange Chromatography

- separation on the basis of the differences in net charges of
Cuts all the AA separately and all AA will be converted to amino acids at different pH conditions with the use of ion-
hydrazide EXCEPT the C-terminal. exchange resins which are either poly-anions or poly-cations.
- applicable to low-molecular-weight polypeptides as well as to
amino acids.
3. Reaction with carboxypeptidase
- will separate the C-terminal from the rest of the peptide chain
A. Cation exchange chromatography
- makes use of a cation exchanger
III. Determine Amino acid sequence
- polysulfonic
- carry fixed negative charges
 Edman’s reaction
- pH gradient used – low pH to high pH
- this reaction liberates amino acids one at a time from the
N-terminus of a polypeptide. i. Fill glass column with polysulfonic exchanger which is
i. The N-terminal residue is removed as a phenylthiohydantoin negatively charged.
derivative. ii. Place the amino acid mixture using a medium with pH 1.
ii. After removal and identification of the N-terminal residue, the iii. The amino acid mixture that you would like to separate is first
new amino-terminal residue so exposed can be identified by dissolved at pH 1 mixture.
repeating the same series of reactions.
iii. This procedure is repeated until the entire sequence is
determined.

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 PROTEIN CHEMISTRY | BIOCHEM A |Sources: Dra. Santos Lecture; LEA THERESE R. PACIS’ trans

At pH 1, what is the charge of the amino acids?  DENATURATION OF PROTEINS

- All amino acids are positively charged
 Basics are +2 DENATURATION
 Neutrals are +1 - loss of three-dimensional structure sufficient to cause loss of function
 Acidics are +1 - occurs when a protein loses its native secondary, tertiary, and/or
quaternary structure. (primary structure is not altered by
- Basics are +2 because there is an amino group at R position denaturation)
which is also positively charged. If the following is destroyed – result:
- In terms of attachment to the column, basics are most attached  Secondary structure - hydrogen bonds between peptide
followed by neutrals then acidics. bonds destroyed  will uncoil.
 Tertiary structure - R to R interaction destroyed  destroys
iv. Wash down the column with a buffer of increasing pH. From globular shape;
pH 1, next, use pH 3, then pH 5, pH 7, then pH 9. As you  Quaternary Structure - between peptide chains  different
increase the pH of the medium, what happens to the amino chains of protein would no longer be connected with one
acids? another – leading to loss of function
- Amino acids become negatively charged.
- Acidics will become negative first because they have the  Denatured state
lowest isoelectric point. - always correlated with the loss of a protein’s function.
- However, loss of protein’s function is not necessarily
- Below IpH is positive, that’s pH 1. Above IpH, they are negative. synonymous with denaturation.
This means that the first to reach zwitterion is the first to - does not necessarily equate with complete unfolding of the
become negative. protein. Most denatured proteins exist in a set of partially folded
states, which as yet are poorly understood.
- Once the acidics become negative, they will be repelled by the - depending on the degree, the molecule may partially or completely
column because it is also negatively charged. Expect acidic lose its biological activity.
amino acids in the first test tube, followed by the neutrals, then
basics. Denaturing agents:
1. Strong acids or bases
B. Anion exchange chromatography 2. Organic solvents
- makes use of an anion exchanger 3. Detergents
- polyamino 4. Reducing agents
- carry fixed positive charges 5. Salt concentration
- pH gradient used – high pH to low pH 6. Heavy metal ions
Start with an exchanger that is positively charged, pH 11. 7. Temperature changes
8. Mechanical stress
At pH 11, what is the charge of the amino acids?
- All amino acids are negative. Changes brought about by denaturation
 Acidics are -2
 Neutrals are -1 A. Chemical alterations
 Basics are -1 - decreased solubility – most visible effect
- many chemical groups which were inactive become exposed and
- Acidics are -2 because they have an extra carboxyl in their R more readily detectable
group.
- The ones that are attached to the column are the acidics because B. Physical alterations
this time the column is positive. - increased viscosity
- decreased rate of diffusion
- As the pH of the solution decreases, the amino acids that started - increased levorotation
as negative will now become positive. - cannot be crystallized
- Which one will become positive first?
 Basics will become positive, followed by the neutrals and then C. Biological alterations
acidics. - increased digestibility
- enzymatic or hormonal activity is destroyed
IV. High Performance Liquid Chromatography (HPLC) - antibody functions are altered

V. ELECTROPHORESIS *Decrease in solubility – most visible effect in globular proteins.

- Separation on the basis of acid-base property of amino acids
facilitated with the use of an electrical field. RENATURATION or REVERSIBLE DENATURATION
i. an ampholyte (protein, peptide or amino acid) in a solution - a process wherein certain globular proteins denatured by heat,
buffered at a particular pH is placed in an electrical field. extremes of pH, or denaturing reagents will regain their native
ii. Depending on the relationship of the buffer pH to the IpH of structure and their biological activity if returned to conditions in
the molecule, the molecule will either move toward the which the native conformation is stable.
cathode (-) or the anode (+), or remain stationary (pH=IpH)
Ex.
- More sophisticated procedures for electrophoresis use polymer  Bovine pancreatic ribonuclease (a digestive enzyme from cattle
gels, starch, or paper as support. that degrades RNA) is denatured when treated with β-
i. inert supports are saturated with buffer solution mercaptoethanol and 8 M urea.
ii. a sample of the proteins or amino acid mixture to be  The enzyme, which is composed of a single polypeptide with four
examined is placed on the support disulfide bridges, completely unfolds and loses all biological activity .
iii. an electric field is applied across the buffered support  Careful removal of the denaturing agents with dialysis results
iv. the proteins migrate in the support towards a charged pole in a spontaneous and correct refolding of the polypeptide and
reformation of the disulfide bonds with full restoration of the
enzyme’s catalytic activity.

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 PROTEIN CHEMISTRY | BIOCHEM A |Sources: Dra. Santos Lecture; LEA THERESE R. PACIS’ trans

*Harper’s Illustrated Biochemistry 30th Edition Table 3-1

18