Chapter 10

PROTEINS

1 Clinical Chemistry I Proteins

 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

2 Clinical Chemistry I Proteins

 Outline
 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

3 Clinical Chemistry I Proteins

 Introduction
 Protein is polymer of amino acids linked covalently
through peptide bonds [polypeptide].
 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

4 Clinical Chemistry I Proteins

 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.

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 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%.

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 Proteins, cont..

 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

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 Protein classification

 Proteins can be classified

based on their composition as:
simple proteins (made from aa only)
• e.g. albumin
complex proteins : (protein + non-protein
parts)
 apoproteins(protein parts)
 conjugated proteins (non-protein parts)
 e.g. Hemoglobin, ferritin, lipoproteins, glycoprotein

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 Protein classification, cont….

Based on their shape as:

Fibrous proteins, e.g. fibrinogen, troponin,
collagen
Globular proteins, e.g. Hgb, enzymes, peptide
hormones, plasma proteins
 Retain their biological activities within narrow
range of T0 and PH.
 When exposed to high temperatures or extremes
of pH causes the molecules of protein to
“denature” and loss their solubility and biological
activities e.g. enzymes lose their catalytic function

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 Protein classification, cont….

 Globular proteins have four levels of structure

 Primary = sequence of amino acid units
 Secondary = folds of the protein strand
 α helix = twisted format
 β sheet = zigzag form
 Tertiary = three dimensional assembly of 20 structure
 Quaternary = association of 2 or more polypeptides

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11 Clinical Chemistry I Proteins
 Properties of protein
 Many properties of proteins are used for their
separation, identification, and assay. The following 5
are among them:

1. Molecular size: most are macromolecules of high

molecular masses, thus separated from smaller
molecules by dialysis, ultrafiltration, molecular gel
filtration chromatography, density gradient
ultracentrifugation

2. Differential solubility: affected by PH, ionic

strength, temp, and dielectric constant of the solvent
12 Clinical Chemistry I Proteins
 Properties cont....
3. Electrical charge: proteins are ampholytes; that is, in
aqueous solutions they may have positive and
negative charges on the same molecule.
 In pH above or below the isoelectric point, proteins become
ionized
 This property is used to separate protein molecules during
electrophoresis.
 The pH of the solution determines the net charge of the molecule
 The pH at which the net charge on the molecules in solution is
zero is called the isoelectric point (pI).
 -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
13 Clinical Chemistry I Proteins
 Properties cont....

4. Adsorption on freely divided inert materials:

these materials offer large surface areas for
interactions [hydrophobic, absorptive, ionic, or
molecular] with proteins
5. Specific binding to antibodies, coenzymes,
or hormone receptors:
 this is the basis for immunochemical assays,
 proteins are also separated by affinity
chromatography, in w/c a ligand attached to a solid
medium provides high selectivity.

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 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
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 Plasma proteins

 Many different proteins are present in the blood, and

collectively known as plasma proteins.
 They include acute phase reaction proteins (APR),
carrier proteins, fibrinogen & other coagulation factors,
complement components, immunoglobulins, enzyme
inhibitors, precursors of substances such as
angiotensin & bradykinin, and many others
 E.g. APR proteins (highly relevant to clinical lab) are
a non-specific response (positively or negatively) to
inflammation (infections, autoimmune diseases, etc)
or tissue damage (trauma, surgery, MI, or tumors)

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  Positive APPs include 1-antitrypsin, 1-acid
glycoproteins, haptoglobulin, ceruloplasmin, C4,
C3, CRP,
 Negative APPs include albumin, transthyretin, and
transferrin.
 Most of plasma proteins are synthesized and
catabolized in the liver.
 g-globulins are made by plasma cells

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 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.

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 Albumin
 the most abundant plasma protein of 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.

 have no carbohydrate side chain but is highly soluble

in water due to of its high net negative charge at
physiological pH
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 Functions of albumin

 maintaining the colloidal osmotic pressure in

both the vascular and extra vascular space with
continuous equilibration between

 binds and transports a large number of cpds,

including free fatty acids, phospholipids, metallic
ions, amino acids, drugs, hormones and bilirubin.

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 Clinical significance of albumin
 Primarily synthesized by the liver and the synthetic
rate may be 300% or more in nephrotic syndrome

 Increased levels of albumin are present only in

acute dehydration and have no clinical
significance.

 Decreased levels are seen in a multitude of clinical

conditions.

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 Clinical Significance cont....
 Decreased levels of albumin is seen in:
 Analbuminemia=
 rare genetic defect with albumin levels less than 0.5g/L clinically
related to abnormal lipid transport
 Inflammatory conditions =
 most common cause, resulting from hemodilution, loss into
extravascular space, increased consumption by cells, decreased
synthesis
 Hepatic disease
 Mostly result from increased Ig levels, loss into EVS, direct
inhibition of synthesis by toxins & alcohols; may happen with
the loss of about 95% of function

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 Clinical Significance cont....
 Urinary loss =
 Normal urine may contain up to 20mg albumin per gram of creatinine.
Excretion above this level suggests either increase molecular filtration,
tubular damage, hematuria, or combination of these.
 Gastrointestinal loss
 Associated with inflammatory disease to GIT, is of little concern
unless the loss is excessive or persists. chronic protein-losing
enteropathy may result in loss similar to nephrotic syndrome.
 Protein energy malnutrition
 Alb levels have been used to help detect and minitor nutritional
status but low levels do not correlate with malnutrition and most
often are due to APR
 Edema and ascites =
 secondary to increased vascular permeability, rather than to
hypoalbuminaemia

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 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.

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 Clinical significance of AFP

 High AFP levels indicates:

 an 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
 Maternal serum levels are decreased in pregnant
women with trisomy 18 or 21

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 C-reactive protein
 Substance present in the sera of acutely ill individuals
that is able to bind the C-polysaccharide on the cell
wall of S.pneumoniae first was described in 1930
 In 1941, it was shown to be protein and given the
name c-reactive protein.
 It is one of the first APPs to become elevated in
inflammatory diseases & also the one exhibiting the
most dramatic increases in concentrations

 consists of five identical subunits and is synthesized

primarily by liver.

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  CRP activates the classical complement path way
starting at C1q and initiates opsonization,
phagocytosis, and lysis of invading organisms such as
bacteria and viruses.

 CRP can recognize potentially toxic autogenous

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

 CRP itself is catabolized after opsonization (make

something susceptible to destruction)

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 Clinical significance

 CRP levels usually rise dramatically after

 myocardial infraction, stress, trauma, infection,
inflammation, surgery, or neoplastic proliferation.
 The increase begins within 6 to 12 hrs of the infraction
& the level may reach 2000 times normal

 Cord blood normally has low CRP concentration,

but in intrauterian infection, the concentration will
be high.

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 CRP

 Determination of CRP is clinically useful for

1. Screening for organic disease
2. Assessment of the activity of inflammatory diseases
3. Detection of inter-current infection in SLE, in
leukemia, or after surgery
4. Management of neonatal septicemia and meningitis,
when specimen collection for bacteriological
investigations may be difficult


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 Analysis of proteins

 Methods used to analyze proteins in body fluids

include
1. Specific quantitative assays of particular proteins
by immunochemical methods using nephlometery,
turbidimetry, radial immunodifusion, RIA, EIA
2. Detection and identification by electrophoresis
3. Quantitative measurement of total protein in
serum, urine , and CSF
4. Analysis by mass spectrometry which provides
structural and quantitative information

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 Quantitative Measurement of Total
Protein in Body Fluids
 When the total protein is measured, two arbitrary
assumptions are made
1. All protein molecules are pure polypeptide chains,
containing 16% by wt of nitrogen
2. Each of the several hundred individual proteins reacts
chemically like every other protein

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 1. Biuret method
 Depends on the presence of peptide bonds
 Peptide bonds react with Cu2+ ions in alkaline
solutions to form a colored product
 The absorbance of w/c is measured
spectrophotometrically at 540nm.
 The buret reagent contains sodium potassium
tartarate to form a complex with cupric acid and
maintain their solubility in alkaline solution. Iodine is
included as an antioxidant

32 Clinical Chemistry I Proteins

 Biuret, cont….
• 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.

33 Clinical Chemistry I Proteins

 Biuret, cont….
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.

34 Clinical Chemistry I Proteins

 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

35 Clinical Chemistry I Proteins

 2. Direct photometric methods.
 Absorption of UV light at 200-225 nm and 272–290
nm is used to measure content of biological
specimens
 Absorption of UV light at 280 nm depend chiefly on
the aromatic rings of tyrosine and tryptophan at PH 8
 Peptide bonds are responsible for UV absorption
(70% at A205) ;
 Specific absorption by proteins at 200 to 225 nm is 10
to 30 times greater than at 280 nm.

36 Clinical Chemistry I Proteins

 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.

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 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. However, a 1:1000
or 1:2000 dilution of serum with sodium
chloride,0.15 mol/l, circumvents this
interferences.

38 Clinical Chemistry I Proteins

 3. Dye-binding methods
 Based on the ability of proteins to bind dyes such as
Amido black 10B and Coomassie Brilliant Blue (CBB).

 The method is simple, easy, and linear up to 150

mg/dL.

 assay of total protein in CSF and urine uses CBB G-

250

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 Specimens

 Urine - Timed
 CSF
 Serum or plasma can not be used due to upper limit
of linearity.

40 Clinical Chemistry I Proteins

 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.

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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

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 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
 Hyporproteinemia: decreased protein due to
burns, renal or intestinal losses, protein energy
malnutrition or sever liver failure.

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 Assay Techniques for serum Albumin
 Dye-binding methods: Bromcresol green (BCG) or
purple (BCP) dyes
 Salt fractionation or the 'salting-out' procedure=
 removal of globulins by salt precipitation followed by the
quantitation of residual albumin in solution by the biuret
or kjeldahl techniques was the standard procedure for
many years
 By difference
 Electrophoresis
 Immunochemical techniques.

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 Serum Albumin BCP Assay Techniques

 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.

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 Serum Albumin BCG Assay Techniques

 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.

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 Specimen For Albumin Testing

 Serum is recommended
 Results tend to be erroneous if the overall serum
protein pattern is abnormal

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 Reference Range
 Adult serum albumin: 3.5 - 5.0 g/dl

 In the upright position levels are about 0.3

g/dl higher because of hemoconcentration.

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 Limitations and Source of Error in serum
Albumin Assay
 Hyperlipemia
 hyperbilirubinemia
 hemolysis
 can generally be eliminated (minimized) by
dilution of serum 1:250

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 Methods for the determination of
total globulins
 Methods for the quantitative determination of
total globulins include
 Colorimetric method
 Globulin by difference
 Electrophoresis: separation of charged molecules
(different proteins) in an electrical field
 Immunochemical technique

50 Clinical Chemistry I Proteins

 Colorimetric method

Test principle:
 In the presence of strong acids, 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

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 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.

52 Clinical Chemistry I Proteins

 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 albumin

53 Clinical Chemistry I Proteins

 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
 Specializedtechniques termed western blotting,
immunofixation, and two-dimensional electrophoresis

54 Clinical Chemistry I Proteins

 Specimen for electrophoresis

 Serum is preferred over plasma since plasma

contains fibrinogen that makes interpretation
difficult.
 CSF
 Concentrated urine

55 Clinical Chemistry I Proteins

 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.

56 Clinical Chemistry I Proteins

 Procedure cont....

 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-

57 Clinical Chemistry I Proteins

 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

58 Clinical Chemistry I Proteins

 Limitations and Sources of Error

 Wrong pH or ionic strength of the buffer

 Wrong voltage
 Too long or too short of time
 Excessive heat

59 Clinical Chemistry I Proteins

 Normal serum protein electrophoresis pattern

Albumin 1 2  

+ -
60 Clinical Chemistry I Proteins
 Serum Protein Electrophoresis:
agarose medium

Cathode: - Electrode
Albumin at bottom
(anodic), than alpha 1,
then alpha 2 then beta 1
and beta 2 then gamma
close to top (cathodic).

Anode: + Electrode
61 Clinical Chemistry I Proteins
 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
1-globulin------------0.2-0.4 g/dl
 2-globulin------------0.4-0.8 g/dl
b-globulin--------------0.5-1.0 g/dl
g-globulin---------------0.6-1.3 g/d

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 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

63 Clinical Chemistry I Proteins

 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.

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 Documentation of protein Results

 Record patient results in result logbook

 Record QC results in QC logbook

 Retain records for recommended time

65 Clinical Chemistry I Proteins

 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
66 Clinical Chemistry I Proteins
 Summary conti…
 Proteins function includes building our body , serving
as enzymes, as antibody. etc..’
 Major plasma proteins include Albumin, Alpha1Acid
lycoproteins, 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
67 Clinical Chemistry I 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.

68 Clinical Chemistry I Proteins

 References
1. Burtis, Carl A., and Ashwood, Edward R.. Tietz:
Fundamentals of Clinical Chemistry. WB Saunders,
Co., Philadelphia, 2001.
2. Arneson, W and J Brickell: Clinical Chemistry: A
Laboratory Perspective 1st ed. FA Davis,
Philadelphia, 2007.
3. Burtis, Carl A., and Ashwood, Edward R.. Tietz:
textbook of Clinical Chemistry. WB Saunders Co.,
Philadelphia, 1999.

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 End of Clinical Chemistry I

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