PROTEINS

PROTEINS
– Most versatile molecules in our bodies
– 10,000-50,000 different proteins exist in our
bodies
 Functions
• Repair body tissues
• Important in blood coagulation and
immunologic function
• For transport of metabolic substances
• Maintenance of osmotic pressure
• Maintenance of blood pH
• Biocatalyst
 AMINO ACIDS
– Large molecules – macromolecules
– Fundamental building blocks of all proteins
– Electric charge: depends on pH of the solution and
type of R group
 CHARACTERISTICS
• Basic solution – (-) charge, proton
dissociate from the carboxyl group present
forming COO
• Acid solution – excess of protons (H+)
which can attach to NH2 group to form
NH3+
• *These charges affect how each protein
moves in an electric field – allows us to
separate proteins
 PROTEIN STRUCTURE
– Determine the shape of a given protein
molecule and affect the function of that
protein
– Peptide Bond (amide linkage)
• Linked together the amino acids of the protein
• Formed when the carboxyl group of one amino acid
joins to the amino group of second forming a
molecule of water as a by product
 Primary Structure
– Number and types of
amino acids
– Amino acid sequence
– Determines the
• identity of proteins
• molecular structure
• binding capacity
• recognition ability
 Secondary Structure
• Coiling and folding of the protein chain
• How the protein chain twists and bends
along its length
– alpha helix – coil, resembling a spring
– beta pleated sheet – flat, corrugated structure
– random – no apparent pattern
 Tertiary Structure
– 3- dimensional shape
– Determine the overall shape of the protein on
how it bends, folds and twists (flat or coils)
– Globular – irregular – ball shaped form twist into
long then rods – fibrous proteins
 Quarternary structure
– When 2 or more polypeptide chains associate
closely together to make-up multichain complex
– Combination of sub-units
 AMINO ACID
ESSENTIAL (supplied through the diet)
▪ Arginine*, Histidine*, Isoleucine, Leucine, Lysine,
Methionine, Phenylalanine, Phenylalanine,
Threonine, Tryptophan, Valine
NON-ESSENTIAL (produced in de novo
synthesis)
 CLASSES OF PROTEINS ACCORDING TO
FUNCTIONS
• Enzymes
– biochemical catalysts
 CLASSES OF PROTEINS ACCORDING TO
FUNCTIONS
• Structural proteins
– Provide structural support for the body, a
tissue or a cell
– Usually long, fibrous molecules
– e.g. collagen – found primarily in the bone
– keratin – hair and nails
 CLASSES OF PROTEINS ACCORDING TO
FUNCTIONS
• Contractile proteins
– Involve in the contraction and relaxation of
muscles
– Usually long, fibrous materials
 CLASSES OF PROTEINS ACCORDING TO
FUNCTIONS
• Antibodies
- neutralize foreign materials
 CLASSES OF PROTEINS ACCORDING TO
FUNCTIONS
• Transport proteins
– Serve a vital function in carrying materials
from one part of the body to another through
circulation
 CLASSES OF PROTEINS ACCORDING TO
FUNCTIONS
• Storage proteins
– Serve as reserves of metal ions and amino
acids
– Ex. Ferritin- for hemoglobin production
 CLASSES OF PROTEINS ACCORDING TO
FUNCTIONS
• Peptide hormones
– Regulate the metabolism, growth and
development, sexual function, reproduction
and behavior
– Chemical Messengers
 CLASSES OF PROTEINS ACCORDING TO
PROTEIN STRUCTURE
• Simple Proteins
– Globular proteins are globe-like,
symmetrical proteins that are soluble in
water.
– Fibrous proteins form long protein
filaments or subunits, are asymmetrical
and usually inert, and are generally
water insoluble due to their hydrophobic
R groups.
 CLASSES OF PROTEINS ACCORDING TO
PROTEIN STRUCTURE
• Conjugated Proteins
– Contains protein and nonprotein
(prosthetic) group
– Examples of conjugated proteins are
• Metalloproteins
• Glycoproteins
• lipoproteins
• nucleoproteins
 SPECIFIC PLASMA PROTEINS
I. Major Components - These are protein
components that are readily detected on
electrophoretic gels stained by conventional
clinical laboratory techniques.
MAJOR COMPONENTS
 A. Prealbumin
• Transthyretin
• It is the fraction that migrates in a position
faster than albumin toward the anode.
• It has a short half--life in the circulation (barely
2 days) compared with other major serum
proteins.
• It is a good indicator for nutritional status of the
body.
• It crosses more easily into the CSF than other
serum proteins.
• Combines with Retinol binding protein to
deliver and transport vitamin A (retinol)
 B. Albumin
1. It is the most abundant protein in the
plasma.
2. It serves as a mobile repository of amino
acids for incorporation into other proteins.
3. It is the general transport protein or
carrier.
4. Its half-life in the circulation is 17-20 days
 B. Albumin
5. Elevations of serum albumin concentration occur in
– Dehydration
– Prolonged application of tourniquet for
venipuncture
6. Measurements of albumin concentrations are
important in interpreting calcium and magnesium
levels because these ions are bound to albumin.
7. Dye Binding methods for albumin measurements
employ
• Bromeresol Green (BCG)
• Hydroxyozobenzene Benzoic Acid (HABA)
8. The presence of albumin in the urine is considered
abnormal even in small amounts.
 Globulin
• With different fraction
▪ Immunoglobulins
▪ Produced and released by plasma cells
▪ Immuno-glubulin G, A, M, D, E
• Reference Values: 2.3 to 3.5 g/dL
 C. 1-antitrypsin
• It’s the major component of the 1-
globulins.
• It has the capacity to combine with trypsin
and inactivate it.
• inhibits of the protease neutrophil elastase.
• It is one of the serum glycoproteins that
rise in response to acute inflammation
• Deficiency in emphysematous pulmonary
disease and juvenile hepatic cirrhosis
• Reference values: 145-270 mg/dL
 D. 2- macroglobulin
1. It is the largest major nonimmunoglobulin
protein in plasma.
2. Women have higher levels than men
because of the hormone estrogen.
3. When other lower molecular weight
proteins are lost, its concentration rises
10-fold or more in nephrotic syndrome.
 β-2 microglobulin
• light chain component of the major
histocompatibility complex (HLA)
• Increased
– inflammatory diseases,
– rheumatoid arthritis
– systemic lupus erythematosus (SLE)
– Renal failure
– Multiple Myeloma
 E. Haptoglobin
1. It is another major protein migrating in the
2 region.
2. It combines with hemoglobin released by
lysed red cells in order to preserve body iron
and protein stores.
3. Its half-life in the circulation is 4 days.
4. It rises in response to stress, infection, acute
inflammation or tissue necrosis
 F. Transferrin
• It is a major B-globulin.
• It transports ferric ions from the iron stores
of intracellular or mucosal ferritin to bone
marrow.
• It is elevated in pregnant women
• Increased in hemochromatosis and IDA
• Dec- liver dxs, malnutrition and nephrotc
syndrome
• Reference values
– 215-365 mg/dL (M)
– 250-380 mg/dL
 G. Fibrinogen
• It is the most abundant of the coagulation
factors.
• It forms the fibrin clot.
• It is elevated with other acute phase
reactants, in pregnancy and the use of
contraceptive medications
• Reference Values
– 200-400 mg/dL
II. MINOR COMPONENTS
 A. Ceruloplasmin
• It is a copper-binding protein.
• It is elevated in individuals under oral
contraceptive therapy and pregnancy.
• It is responsible in oxidizing iron from
ferrous to ferric.
• Disordered copper metabolism results to
Wilson's disease due to ceruloplasmin
deficiency.
• Features of Wilson’s Disease:
– Deposition of copper in skin, liver and cornea
(Kayser-Fleischer ring)
 B. Hemopexin
• It binds heme released by degradation of
hemoglobin.
• It is most profoundly decreased in
intravascular hemolysis
• Migrates in the ß-region during
electrophoresis
• Reference values: 50-115 mg/dL
 C. 1-Acid Glycoprotein
• It is also known as orosomucoid.
• It is elevated during pregnancy,
• It binds to progesterone and could be
important in its transport or metabolism
• Low pH, negatively charged
• Reference values: 50-120 mg/dL
 D. C-Reactive Protein
• It is present in serum of patients with disorders
other than pneumococcal infections and tissue
necrosis.
• It serves as a general scavenger molecule.
• CRP is the earliest indicator of inflammation
• CRP levels are sometimes used as a rapid test for
presumptive diagnosis of bacterial infection (high
CRP) versus viral infection (low CRP).
• It is used to monitor the progression or remission
of autoimmune disease.
• Persons with high normal CRP concentrations are
at greater risk for stroke or myocardial infarction
than those with low normal values
 E. Alpha- I - fetoprotein (AFP)
• AFP is increased in amniotic fluid and serum in
neural tube defects (spina bifida).
• Decreased level is associated with Down's
syndrome
• A tumor marker for hepatic and gonadal cancer
• Reference values: 5 ng/mL
• Methods: RIA, EIA and Affinity Chromatography
based on reactivity to Lens culinaris agglutinin
AFP L3
METHODS OF DETERMINATION
Total Protein Determination
Albumin Determination
 TECHNIQUES OF PROTEIN SEPARATION
Electrophoresis may be divided into:
• Moving boundary or frontal electrophoresis
a. At pH 7.6, four serum protein fractions
(albumin, alpha, beta, gamma) are
identified and quantified optically by
change in refractive index at the boundaries
among these bands.
b. Separation is achieved in a homogeneous
solution without solid support medium thus
convective forces prevented resolution into
distinct zones.
Standard dyes used in Electrophoresis
• a. For serum electrophoresis
– Coomasie Brilliant Blue
– Ponceau S
– Amido Black
• b. For lipoproteins
– Oil Red 0
– Sudan Black
• c. For glycoproteins
– Periodic Acid Schiff
Electrophoretic Pattern
Electrophoretic Pattern
Electrophoretic Pattern
 PROTEIN DETECTION AND QUANTITATION

• Kjeldahl Technique (Reference Method)

– This technique consists of an acid digestion to
release ammonium ions from nitrogen-
containing compounds.
 Kjeldahl Technique (Reference Method)

• The ammonium can then be quantitated

by conversion to ammonia gas and
titration as a base or by Nesslerization.
• Total serum protein is obtained by
multiplying the value of total protein
nitrogen (TPN) by 6.25.
 Refractive Index
• Is accurate for measuring serum protein
concentration as dissolved solute for
levels above 2.5 g/dL.
• It cannot be used for urine protein
measurements because of excess
amounts of solutes in relation to the
protein.
 Turbidimetric method
• They are used to measure protein concentration
in CSF or urine.
• Protein forms precipitate in the addition of
trichloroacetic acid, sulfosalicylic acid or other
acid reagent.
• Turbidity produced can be measured by optical
density.
• They are not specific to proteins because other
acid insoluble substances such as nucleic, acids
can also precipitate,
 Appearance Protein Content

Negative Clear sample.No <20 mg/dL

turbidity
Trace Very Faint 20-200 mg/dL
Precipitate
1+ Small degree of 100-1000 mg/dL
turbidity
2+ Moderate turbidity 1000-2500 mg/dL

3+ Heavy turbidity 2500-4500 mg/dL

4+ Heavy flocculation/ >4500 mg/dl

clumping
COLORIMETRIC TECHNIQUE
 Biuret Method
• It is highly specific for proteins and peptides.
• it depends on the presence of two or more
peptide bonds which-form a purple complex
with copper, salts in alkaline solution.
• Ammonium ion, Hb and bilirubin can cause
interferences,
• Reagents:
– Copper sulfate
– Tartrate salt
– Potassium iodide
 Clinical Significance
Parameter Reference Increased Values Decreased Values
Range
Total Protein 6.5-8.5 g/dL Dehydration GI Cancers
Severe Exercise Liver Disease
Infection Malnutrition
Cancer Low Thiamine
Glomerulonephritis
 Clinical Significance
Parameter Reference Increased Decreased
Range Values Values
Albumin 3.5-5.0 g/dL Dehydration Pregnancy
Sunstroke Malnutrition
Exercise Malabsorption
Multiple Liver disease
Sclerosis Kidney disease
Hypothyroidis Burns
m