CHAPTER 10

PROTEINS
 Acknowledgements
• Addisa Ababa University
• Jimma University
• Hawassa University
• Haramaya University
• University of Gondar
• American Society for Clinical Pathology
• Center for Disease Control and Prevention-
Ethiopia
 Objectives

At the end of this chapter, the student is expected to:

• Describe basic protein structure and composition

• Define amino acids
• State and describe the four stages of protein structure
• Show how protein properties can be used for their
identification and assay
• List the proteins synthesized by the liver
• Describe the physiological functions of proteins
• Describe how plasma protein abnormalities reflect
severity of hepatic dysfunction
• Describe the methods used for determination of protein
concentration in blood
 Outline of protein lecture
• Introduction
• Classification of proteins
• Functions of proteins
• The plasma proteins
• Plasma proteins with clinical significance
• Properties of proteins
• Specific methods for the determination of proteins
• Serum protein electrophoresis
• Samples
• Interpretation
• Quality control
• summary
 Introduction

 Proteins- amino acids polymers linked covalently

through peptide bonds.
 Amino acid: an organic compound containing
both amino and carboxyl functional groups;
simplest units of proteins
 There are 20 different kinds of amino acids,
combined in different proportion and
arrangements to build all protein molecules
 Amino Acids
• Two amino acids combined by peptide
bond are called a dipeptide.
• When amino acids involved in the bond
formation become 3, 4, 5 they are named
as tri-, tetra-, and penta- peptides
respectively.
 Proteins
 All proteins contain carbon, hydrogen, oxygen,
and nitrogen.
 Some proteins may also contain sulfur
phosphorous, copper, iron, zinc, iodine, and
other elements.
 The presence of nitrogen in all proteins sets
them apart from carbohydrates and lipids.
 The average nitrogen content of proteins is
approximately 16%.
 Proteins, continued..
 Comprises 50-70% of cell’s dry weight
 Found in cells, as well as in all fluids,
secretions, and excretions
 More than 300 different types of plasma
proteins have been discovered
 Protein classification
Proteins may classified based on their
composition as:
• simple proteins
- albumin

• complex proteins :
- apoproteins,
- conjugated proteins
 Protein classification, continued….

• Based on their shape proteins can be classified

as fibrous proteins and globular proteins
• Proteins have four levels of structure
-Primary structure
-Secondary structure
-Tertiary structure
-Quaternary structures.
 Proteins as ampholytes
• In pH above or below the isoelectric point,
proteins become ionized
– --COOH –alkaline pH  COO- anionic
– --NH2 –acidic pH  NH3+ cationic
– H2N-R-COOH --neutral pH ampholyte
– Both COO- and NH3+ exist so net charge is neutral
 Function of Proteins

• used to construct or build our body

• catalyze biochemical reactions as an enzyme
• regulate body metabolism as hormones
• protect our body from foreign body attack as an antibody
and components of complement
• maintain osmotic pressure in plasma
• transport different lipids, minerals, hormones, vitamins
etc as hemoglobin, apolipoprotein, albumin etc
• assist to arrest bleeding and maintain homeostasis as
coagulation factor
 Plasma proteins

• Many different proteins are present in the blood,

and collectively known as plasma proteins.
• They include Albumin, Alpha1Acid
glycoproteins, ceruloplasmin, C-reactive protein,
complements, fibrinogen and immunoglobulins.
• Most of plasma proteins are synthesized and
catabolized in the liver.
• -Globulins are made by plasma
cells
 Clinical significance of protein
• The two general causes of alteration of serum
total protein are:
change in volume of plasma water
change in concentration of protein
The relative hypoproteinemia --hemodilution.
The relative hyperproteinemia-hemoconcentration.
 Albumin

• the most abundant plasma protein extra

vascular body fluids, including CSF, Interstitial
fluid, urine, and amniotic fluid.
• accounts approximately one-half of the plasma
protein mass.
• a globular protein, with molecular mass of 66.3
KD.
• Because of its high net negative charge at
physiological pH, highly soluble in water, but
does not have carbohydrate side chain.
 Functions of albumin

• maintaining the colloidal osmotic pressure in

both the vascular and extra vascular space with
continuous equilibration in between
• binding and transportation of large number of
compounds, including free fatty acids,
phospholipids, metallic ions, amino acids, drugs,
hormones and bilirubin.
 Clinical significance

Cause for an increase level of albumin

• acute dehydration
• Increased production of albumin has no clinical
significance.
 Clinical Signficance

Decreased levels of albumin seen in:

• Edema and ascites
• Analbuminemia
• Urinary loss
• Inflammatory conditions
• Gastrointestinal loss
• Hepatic disease
• Protein energy malnutrition
 Alpha 1 –fetoprotein [AFP]

• one of the first α –globulin appear in mammalian

sera during development of the embryo
• dominant serum protein in early embryonic life
• synthesized primarily by the fetal yolk sac and
liver..
• contains approximately 4% carbohydrate with a
molecular mass approximately 70KD.
 Clinical significance
High AFP levels seen in:
• open neural tube or abdominal wall defect in
fetus.
• Multiple fetuses,
• fetal demise,
• fetomaternal bleeding, and
• incorrect estimation of gestational age
Hepatocellular and germ cell carcinomas in
childhood and adults
 C-reactive protein

• The first APPs to become elevated in

inflammatory diseases
• consists of five identical subunits and is
synthesized primarily by liver.
• C-reactive protein (CRP) found in sera of acutely
ill individuals from S. pneumonia
• CRP activates the classic complement path way
starting at C1q and initiates opsonization,
phagocytosis, and lysis of invading organisms.
such as bacteria and viruses.
 CRP continued…

• CRP can recognize potentially toxic autogenous

substances released from damaged tissue, to
bind them, and then detoxify or clear them from
the blood.
 Clinical significance

CRP levels usually rise after

• myocardial infraction, stress, trauma, infection,
inflammation, surgery, or neoplastic proliferation.

CRP is clinically useful for

• Screening for organic disease
• Assessment of the activity of inflammatory
diseases
 CRP
• Detection of inter-current infection in systemic
lupus erythematosus (ALE), in leukemia, or after
surgery\

• Management of neonatal septicemia and

meningitis

• Cord blood normally has low CRP concentration,

but in intrauterian infection, the concentration will
be high.
Methods based on Properties of Proteins

• Molecular size
• Differential solubility
• Electrical charge
• Adsorption on finely divided inert materials
• Specific binding to antibodies, coenzymes, or
hormone receptors
 Specific methods for total protein
determination.
• Biuret method
• Direct photometric methods.
• Dye-binding methods.
• Turbidimetric and nephelometric methods
 Biuret method

Principle of the test

• peptide bonds react with Cu2+ ions in alkaline
solutions to form a colored product
• absorbance is measured spectrophotometrically
at 540nm.
 Biuret, continued….

• One copper ion probably is linked to 6 nearby

peptide linkage by coordinate bonds.
• Amino acids and di peptides do not react, but tri
peptides, oligo peptides, and polypeptides react
to yield pink to reddish- violet products.
• The intensity of the color produced is
proportional to the amount of protein present in
the reaction system.
 Biuret, continued….

Specimen type and preservation

 Either serum or plasma, but serum is preferred.
 A fasting specimen may be required to decrease the
risk of lipemia.
 Hemolysis should be avoided.
 Serum samples are stable for at least 1week at room
temperature and for 1 month at 2 to 4o C.
 Specimens that have been frozen and thawed should
be mixed thoroughly before assay.
 Limitations and Sources of Error
• The biuret reaction occurs with other compounds
with structural similarity.
• Hemolysis
• Lipemia
• Ammonium ions interfere
• Sensitivity range in the g/dL so suitable for
serum specimens but not other body fluids
 Direct photometric methods.
Principle of the test.
 Absorption of UV light at 200-225 nm and 272 –
290 nm is used
 Absorption of UV light at 280 nm depend on the
aromatic rings of tyrosine and tryptophan
 Peptide bonds are responsible for UV absorption
(70% at A205) ;
 Specific absorption by proteins at 200 to 225 nm
10 to 30 times greater than at 280 nm.
 Specimen
 Either serum or plasma, but serum is
preferred.
 A fasting specimen may be required to
decrease the risk of lipemia.
 Hemolysis should be avoided.
 Limitations and Sources of Error

Accuracy & specificity suffer from

 uneven distribution of tyrosine and tryptophan
among individual proteins
 the presence of free tyrosine and tryptophan,
uric acid, and bilirubin, which also absorb light
near 280nm.
 interferences from free tyrosine and tryptophan
is significant at 200 to 225nm.
 A 1:1000 or 1:2000 dilution of serum with
sodium chloride,0.15 mol/l ,circumvents this
interferences.
 Dye-binding methods

Principle of the test

 Based on the ability of proteins to bind dyes
such as amido black 10B and Coomassie
Brilliant Blue.
 The method is simple, easy, and linear up to 150
mg/dL.
 assay of total protein in CSF and urine uses
CBB G-250
 Specimens
• Urine
– Timed
• CSF
• Serum or plasma can not be used due to
upper limit of linearity.
 Limitations or Sources of Error

Dye binding methods

 unequal affinities and binding capacities of
individual proteins for dyes
 inability to define a consistent material for use
as a calibrator.
 Turbidimetric and nephelometric
methods
Principle of the test
 Protein in the sample is precipitated with
addition of sulfosalicylic acid alone, with
sulfosalicylic acid in combination with sodium
sulfate or trichloroacetic acid (TCA), or with TCA
alone to produce turbidity.
 Degree of turbidity measured with
Turbidometeric or nephelometric methods
 Reference Ranges and
Interpretation
• Serum total protein: 6-8 g/dL

• Hyperproteinemia: increased serum total

protein due to dehydration or increased
gamma globulins such as in multiple myeloma
• Hyperproteinemia: decreased protein due to
burns, renal or intestinal losses, protein
energy malnutrition or sever liver failure.
 Quality Control
• A normal & abnormal quality control sample
should be analyzed along with patient samples,
using Westgard or other quality control rules for
acceptance or rejection of the analytical run.
– Assayed known samples
– Commercially manufactured (Humastar)

• Validate patient results

• Detects analytical errors.
Documentation of Protein Results
• Record patient results in result logbook
• Record QC results in QC logbook
• Retain records for recommended time
 Assay Techniques for serum Albumin

 Dye-binding methods: BCP and BCG

 Salt fractionation or the 'salting-out'
procedure
 By difference
 Electrophoresis
 Immunochemical techniques.
 Serum Albumin Assay Techniques
BCP Method
Test principle:
 Yellow BCP dye, buffered at pH 5.2 with
acetate
 turns green when complexed with albumin.
 Absorbance of the green complex is
measured at 603 nm.
 Dye binding method for serum Albumin

BCG Method
 Test principle: Albumin and BCG are allowed to bind
at pH 4.2, in succinate buffer,
 absorption of the BCG-albumin complex is measured
at 628 nm.
 At pH 4.2, albumin acts as a cation to bind the
anionic dye.
 The reaction is extremely fast and goes to
completion in only a few seconds.
 Specimen

 Serum is recommended
 Results tend to be erroneous if the overall
serum protein pattern is abnormal
 Reference Range

Adult serum albumin

 Recumbent: 3.5 - 5.0 g/dl

 In the upright position levels are about 0.3 g/dl

higher because of hemoconcentration.
Limitations and Source of Error in serum
Albumin Assay

 hyperlipemia
 hyperbilirubinemia
 hemolysis
 can generally be eliminated (minimized) by
dilution of serum 1:250
 Quality Control
• A normal & abnormal quality control sample
should be analyzed along with patient samples,
using Westgard or other quality control rules for
acceptance or rejection of the analytical run.
– Assayed known samples
– Commercially manufactured (Humastar)

• Validate patient results

• Detects analytical errors.
Documentation of Albumin Results
• Record patient results in result logbook
• Record QC results in QC logbook
• Retain records for recommended time
 Methods for the determination of
total globulins
Methods for the quantitative determination of
total globulins
 Colorimetric method
 Globulin by difference
 Electrophoresis: separation of charged
molecules (different proteins) in an electrical
field
 Immunochemical technique
 Colorimetric method

Test principle:
 Glyoxylic acid reacts with tryptophan residues
of proteins to form a purple color.
 Copper sulfate is added to enhance color
formation.
 human globulins are known to contain 2 - 3%
tryptophan
 Protein Electrophoresis
• Principle: The pH of the solution determines
the net charge of the protein molecules.
• At pH 8.6, hydrogen ions will be lost from the
carboxyl ends and from functional groups of R
residues of the amino acids.
• Since proteins are composed of different
amino acids, when voltage is applied, they
migrate to different positions on the cellulose
or agarose media.
 Materials and procedures of protein
electrophoresis

 Buffer: barbital with an ionic strength of 0.05 and

pH 8.6
 Sample volume: 3 to 5 µl
 Power supply: 1.5 mA per 2-cm width of cellulose
acetate medium; 10mA per 1-cm width of agarose
medium
 Run time:40 to 60 min producing a 5- to 6-cm
migration distance for aalbumin
 Serum Protein Electrophoresis

• Electrophoresis is widely used in clinical

laboratories to study and measure the protein
content of biological fluids- serum, urine or csf.
• Screening tool for protein abnormalities
• Electrophoresis techniques include:
– Cellulose acetate electrophoresis
– Gel and capillary electrophoresis
– Specialized techniques termed western blotting,
immunofixation, and two-dimensional
electrophoresis
 Specimen for electrophoresis
• Serum
• CSF
• Concentrated urine
 Procedure for Protein
Electrophoresis
 Patient’s specimen is placed into a sample
trough within agarose gel, is placed in an
alkaline buffer solution
 a standardized voltage is applied and allowed to
run for 1hr
 the agarose gel is processed in acetic acid and
alcohol washes to fix the proteins in the
agarose.
 the protein fractions are stained with Coomassie
Brilliant Blue protein stain.
 Procedure for Protein
Electrophoresis
 After a second wash, fixed protein bands
can be visualized and quantified with
densitometry.
 In normal serum electrophoresis 5-6
bands are visible:
 Albumin
 Globulins: α1-, α2-, β-, and γ-
 Electrophoresis Calculations
• Total serum protein x % fraction gives quantity in
g/dL
• Example: TSP 6.0 g/dL and % albumin of 50%
albumin = 6.0 x 50% = 3.0 g/dL
• TSP – Albumin = globulins
• Example TSP 6.5 g/dL and albumin 3.5 g/dL
• Globulins = 6.5 – 3.5 = 3.0 g/dL
• Albumin/ globulin ratio
• Example 3.5/ 3.0 = 1.2
 Limitations and Sources of Error
• Wrong pH or ionic strength of the buffer
• Wrong voltage
• Too long or too short of time
• Excessive heat
 Normal serum protein electrophoresis pattern

Albumin 1 2  

+ -
Serum Protein Electrophoresis:
agarose medium
Cathode: - Electrode

Anode: + Electrode
 Reference Ranges

 Reference Range of total protein

– Serum---------------------------6-8 g/dl
– CSF----------------------------- 8-32 mg/dl
 For electrophoresis
-serum: albumin-----------------3.9-5.1 g/dl
α1-globulin------------0.2-0.4 g/dl
α2-globulin------------0.4-0.8 g/dl
β-globulin--------------0.5-1.0 g/dl
γ-globulin---------------0.6-1.3 g/d
 Interpretation of Protein
Electrophoresis Results
• Further resolves cause of hyperproteinemia
– Gamma globulin increase
• Multiple myeloma
– Normal gamma globulins but increased albumin
• Hemoconcentration
• Look for individual increases in alpha or beta
globulins
 Quality Control
 A normal & abnormal quality control sample should
be analyzed along with patient samples, using
Westgard or other quality control rules for
acceptance or rejection of the analytical run.
– Assayed known samples
– Commercially manufactured
 Validate patient results
 Detects analytical errors.
 Documentation of protein
Results

• Record patient results in result logbook

• Record QC results in QC logbook
• Retain records for recommended time
 Summary

 Proteins are polymers of amino acids that are linked covalently

through peptide bonds.

 The presence of nitrogen in all proteins sets them apart from

carbohydrates and lipids.

 Proteins are classified based on the number of amino acid

molecules,composition of amino acids.

 Protein have four structural levels;10,20,30,and 40.

 Properties of proteins include molecular size, differential

solubility, electrical charge, adsorption on finely divided inert
materials, and specific binding to antibodies, coenzymes, or
hormone receptors
 Summary, continued…
• Proteins function includes building our body , serving as enzymes, as
antibody. etc..’

 Major plasma proteins include Albumin, Alpha1Acid glycoproteins,

ceruloplasmin, C-reactive protein, complements, fibrinogen and
immunoglobulins

 Increase level of protein caused by acute dehydration and has no

clinical significance; decreased levels of proteins seen in edema and
ascites, analbuminemia, urinary loss, inflammatory conditions,
gastrointestinal loss, hepatic disease, protein energy malnutrition.

 Specific methods for total protein determination include Biuret method,

direct photometric methods, dye-binding methods, turbidimetric and
nephelometric methods

 Serum protein electrophoresis used to fractionate proteins

 Review Questions
• What properties of proteins allow for
electrophoresis?
• Why are different methods used for
measuring total protein in serum than in
urine?
• What are two causes of hyperproteinemia?
• Describe how liver disease or kidney disease
can affect serum albumin and protein levels.
 Reference

1. Burtis, Carl A., and Ashwood, Edward R.. Tietz:

Fundamentals of Clinical Chemistry. Philadelphia,
2001.

2. Arneson, W and J Brickell: Clinical Chemistry:

A Laboratory Perspective 1st ed. 2007 FA Davis

3. Burtis, Carl A., and Ashwood, Edward R.. Tietz:

textbook of Clinical Chemistry. Philadelphia, 1999.
End of Clinical Chemistry I