PROTEINS

EARL ADRIANE A. CANO, RMT

CLINICAL CHEMISTRY LABORATORY
 IMPORTANCE OF
PROTEINS
• Energy production
• Water Distribution
• Transporter
• Antibodies
• Structural Proteins
• Cellular Hormones
• Enzymes

EARL ADRIANE A. CANO, RMT -ENOtes :)

 AMINO ACIDS

EARL ADRIANE A. CANO, RMT -ENOtes :)

 PROTEINS ARE
MACROMOLECULES

Simple vs. Conjugated

EARL ADRIANE A. CANO, RMT -ENOtes :)

 SYNTHESIS
• Synthesized in the liver
• Secreted by the hepatocyte into the circulation
• Each protein has its own unique amino acid sequence

EARL ADRIANE A. CANO, RMT -ENOtes :)

 STRUCTURE

• Primary Structure
• Secondary Structure
• Tertiary Structure
• Quaternary Structure

EARL ADRIANE A. CANO, RMT -ENOtes :)

 PROTEIN PROFILE
• TOTAL PROTEIN TOTAL PROTEIN NORMAL
• ALBUMIN DETERMINATION VALUES:

• Na2SO4 •6.6-8.7 mg/dL (S.I. Unit)

•66-87 g/L (Conventional)
• Na2SO3
• (NH)2SO4
ALBUMIN NORMAL VALUES:
• Methanol
• 3.8-5.1 g/dL (S.I. Unit)
• GLOBULIN DETERMINATION • 38-51 g/L (Conventional)
• ALBUMIN-GLOBULIN RATIO (A/G)
𝟏.𝟓−𝟑.𝟓
• Normal A/G =
𝟏

EARL ADRIANE A. CANO, RMT -ENOtes :)

 PREALBUMIN
• Transport system for T3 and T4
• Decreased in hepatic damage, acute-phase
inflammatory response, and tissue necrosis
• low prealbumin level

EARL ADRIANE A. CANO, RMT -ENOtes :)

 ALBUMIN
• Fastest migrating protein
• Most dense but with lowest Molecular Weight
• Purely synthesized in the liver from 585 amino acids
• Protein present in highest concentration in the plasma
• Responsible for nearly 80% of the colloid osmotic
pressure of the intravascular fluid
• buffers pH and negative acute-phase reactant protein
• capacity to bind various substances in the blood

EARL ADRIANE A. CANO, RMT -ENOtes :)

• Decreased concentrations of serum albumin may
be caused by the following:
• Inadequate source of amino acids
• Liver disease
• Protein-losing enteropathy or gastrointestinal
loss as in diarrhea
• Kidney loss to the urine in renal disease
• Skin loss in the absence of skin barriers (i.e.
burns)
• Hypothyroidism
• Dilution by excess (polydypsia or excess
administration of IV fluids)
• Acute disease states
• Mutations: albuminemia &Bisalbuminemia
• Sepsis
EARL ADRIANE A. CANO, RMT -ENOtes :)
 GLOBULINS
• Has four fractions: α1, α2, β, γ

EARL ADRIANE A. CANO, RMT -ENOtes :)

 Α1 FRACTION GLOBULINS:
•  α1-ANTITRYPSIN
• Most important function: inhibition of neutrophil elastase
•  α1-FETOPROTEIN
• Synthesized in the developing embryo and fetus and
then by the parenchymal cells of the liver
•  α1-ACID GLYCOPROTEIN
• Major plasma glycoprotein
•  α1-ANTICHYMOTRYPSIN
• Acute-phase protein that is increased during
inflammation
• Inhibits the activity of the enzymes cathespin G,
pancreatic elastase, most cell chymase, and
chymotrypsin
EARL ADRIANE A. CANO, RMT -ENOtes :)
•  INTER-α-TRYPSIN INHIBITOR
• A family of serine protease inhibitors, assembled from two
precursor proteins: a light chain (bikunin) and either one
or two heavy chains

•  GC-GLOBULIN
• Group-specific component; Vitamin D-binding protein

EARL ADRIANE A. CANO, RMT -ENOtes :)

 Α2 FRACTION GLOBULINS:
•  HAPTOGLOBIN
• α2 glycoprotein ; synthesized in the hepatocytes
• tetramer; consisting of two α and two β chains
• bind free hemoglobin to prevent the loss of hemoglobin
and its constituent, iron, into the urine
• evaluate hemolytic anemia and to distinguish it from
anemia due to other causes

EARL ADRIANE A. CANO, RMT -ENOtes :)

•  CERULOPLASMIN
• Copper-containing, α2 glycoprotein enzyme that is
synthesized in the liver
• Wilson’s disease
•  α2-MACROGLOBULIN
• Largest protein in serum
• Synthesized in the liver

EARL ADRIANE A. CANO, RMT -ENOtes :)

 Β FRACTION GLOBULINS:
•  TRANSFERRIN (Siderophilin)
• Major component of the β globulin fraction and appears
as a distinct band on high resolution serum protein
electrophoresis
•  HEMOPEXIN
• Function of hemopexin is to scavenge the heme
released or lost by the turnover of heme proteins
•  LIPOPROTEINS
• Complexes of proteins and lipids whose function is to
transport cholesterol, triglyceride, and phospholipids in
the blood
•  β2- MICROGLOBULIN
• Smallest protein in serum
• Light chain component of the major histocompatibility
complex (HLA)

EARL ADRIANE A. CANO, RMT -ENOtes :)

•  COMPLEMENT
• One of the natural defense mechanisms against
infections
•  FIBRINOGEN
• One of the largest proteins in blood plasma
• On plasma electrophoresis, fibrinogen is seen as a distinct
band between the β and γ globulins
•  C-REACTIVE PROTEIN
• One of the first acute-phase proteins to rise in response to
inflammatory disease
•  HIGH SENSITYIVITY C-REACTIVE PROTEIN
• same protein but is named for the newer, monoclonal
antibody-based test methodologies that can detect CPR
levels below 1mg/dL

EARL ADRIANE A. CANO, RMT -ENOtes :)

 γ
FRACTION GLOBULINS:

•  IMMUNOGLOBULINS
• antibodies
• synthesized in spleen (secondary lymphoid organs)
• consists of two identical heavy and two identical light
chains (either κ or λ)
• five classes of immunoglobulins:
• Immunoglobulin G (IgG)
• Immunoglobulin A (IgA)
• Immunoglobulin M (IgM)
• Immunoglobulin E (IgE)
• Immunoglobulin D (IgD)

EARL ADRIANE A. CANO, RMT -ENOtes :)

 PROTEIN MEASUREMENT
• DIRECT METHOD

• Biuret Reagent Contains:

• CuSO4 in NaOH
• Rochelle Salt: Potassium Sodium Tartrate
• Potassium Iodide

EARL ADRIANE A. CANO, RMT -ENOtes :)

• INDIRECT METHOD
• Proteins are 16% Nitrogen; 100 ÷ 16 =6.25 (factor)
• Kjeldahl-Nessler Method
• Kjeldahl Digestion
• 1st Nessler’s Reaction
• 2nd Nessler’s Reaction

EARL ADRIANE A. CANO, RMT -ENOtes :)

 DYE BINDING METHOD
*Dyes for Protein Bonds:
• Ponceau S
• Bromphenol Blue
• Amido Black
• Silver Stain
• Coomassie Brilliant Blue
• Bromcresol Green
• Hydroxyazobenzoic acid (HABA)

EARL ADRIANE A. CANO, RMT -ENOtes :)

 OTHER METHODS
(ALTERNATIVES TO DIRECT
METHOD/BIURET REACTION)
• Folin-Ciocalteu/Gallic Acid Equivalence (GAE) Method
• Phosphotungstomolybdic acid solution
• Chromogens: Tungsten Blue and Molybdenum Blue

• Ninhydrin Reaction
• Chromogens: Ruhemann’s Purple

• TURBIDIMETRIC METHODS
• For Urine and CSF Protein analyses
• Reagent: Trichloroacetic Acid (TCA) or Sulfsalicylic acid
(SSA)

EARL ADRIANE A. CANO, RMT -ENOtes :)

 SEPARATION OF PROTEIN
AND ITS COMPONENTS
• CHROMATOGRAPHY
• Used in analyzing various amino acids

𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒

𝑹𝒇 𝑽𝒂𝒍𝒖𝒆 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 (𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡)
EARL ADRIANE A. CANO, RMT -ENOtes :)
• ELECTROPHORESIS
• *SPE – Serum Protein Electrophoresis

•  Moving Boundary Electrophoresis/Frontal

Electrophoresis

• Zonal Electrophoresis
• Cellulose acetate
• Agarose
• Polyacrylamide gel

EARL ADRIANE A. CANO, RMT -ENOtes :)

EARL ADRIANE A. CANO, RMT -ENOtes :)
EARL ADRIANE A. CANO, RMT -ENOtes :)
 PROTEIN MARKERS
• BJP (Bence-Jones Protein)
• CEA (Carcinoembryonic Antigen)
• AFP (α1-Fetoprotein)
• CA 125
• CA 15-3
• CA 19-9
• PSA (Prostate Specific Antigen)
• hCG (Human Chorionic Gonadotropin)
• βhCG

EARL ADRIANE A. CANO, RMT -ENOtes :)

 HYPOPROTEINEMIA
• A total protein level less than the reference
interval, occurs in any condition where a negative
nitrogen balance exists
• Caused by:
• Excessive loss
• Decreased intake
• Decreased protein synthesis in liver disease or in inherited
immunodeficiency disorders
• Accelerated catabolism of proteins

EARL ADRIANE A. CANO, RMT -ENOtes :)

 HYPERPROTEINEMIA
• Increase in total plasma proteins; not an actual disease
state but is the result of an underlying cause,
dehydration
• Although the absolute quantity of proteins remains
unchanged, the concentration is elevated due to a
decreased volume of solvent water

EARL ADRIANE A. CANO, RMT -ENOtes :)

EARL ADRIANE A. CANO, RMT -ENOtes :)