PROTEINS 3.

Tertiary Structure
• Large molecules (macromolecules) • Refers to the way in which the twisted chain folds back on itself to form the
• Composed of fundamental building blocks called amino acids three dimensional structure
• Basic components of AA: • Responsible for many of the physical and chemical properties of the protein
➢ Amino group NH 4. Quarternary Structure
➢ Carboxylic group COOH • 2 or more polypeptide chains
➢ Carbon skeleton • Chains → monomers or subunits
• Ex. Hemoglobin → comprises 4 globin chains
• R → stands for a number of groups ranging from a simple H to a much more Lactate dehydrogenase → 5 peptide chain
complex phenolic derivative Creatine kinase → 2 peptide chains
• AA are linked to each other through peptide bonds – the covalent peptide bond is
formed when the carboxyl group of one AA joins the amino group of another Denaturation:
• Disruption of the bonds can cause inactivation or loss of function of the protein
Nitrogen Content: • Can be caused by heat, hydrolysis by strong acid or alkali, enzymatic action,
• Comprise the elements carbon, oxygen, hydrogen, nitrogen and sulfur exposure to urea or other substances, exposure to UV light, mechanical forces
• Average – 16% (vigorous mixing)

Charge and Isoelectric Point: Classification Of Proteins (based on composition):

• Amphoteric (positive and negative charges) – depending on the pH of their 1. Simple Proteins
environment • Contain peptide chains that on hydrolysis yield only AA
• In highly acidic solutions (low pH) → AA have a net (+) charge • May be globular or fibrous in shape
• In highly alkaline solutions (high pH) → AA have a net (-) charge • Globular → ex. albumin
• Each AA has a pH at which it is neutral or no net surface charge → isoelectric • Fibrous → collagen and troponin
point (pI) 2. Conjugated Proteins
• Comprise a protein (apoprotein) and a nonprotein moiety (prosthetic group)
Shape: • The prosthetic group may be lipid, carbohydrate, porphyrins, metals, etc.
• Globular and fibrous • Ex. metalloproteins → have a metal ion attached to the protein (ferritin,
• Globular → compact, tightly folded and coiled chains; majority of serum proteins ceruloplasmin)
are globular lipoproteins → cholesterol and triglycerides
• Fibrous → hair, collagen, and fibrin (structural proteins) glycoproteins → carbohydrates
mucoproteins/proteoglycans → heparan, keratin, chondroitin sulfate
Solubility:
• Fibrous proteins are insoluble in aqueous solution General Functions of Proteins:
• Globular proteins are generally soluble in water or weak salt solutions • Tissue maintenance
• properties of the solute that influence solubility: • Maintenance of water distribution between cells and tissue, interstitial
✓ pH compartments and the vascular system of the body
✓ ionic strength • Participation as buffers to maintain pH
✓ temperature • Transportation of metabolic substances, examples:
✓ dielectric constant ✓ thyroxine binding globulin (TBG) carries thyroxine
✓ haptoglobin binds free hemoglobin
Immunogenicity: ✓ albumin transport free fatty acids
• effective antigens (tyrosine) ✓ unconjugated bilirubin, etc.
• Part of defense system (antibodies) → help protect the body against infection
Protein Structure: • Hormones and receptors, examples:
1. Primary Structure ✓ ACTH
• The number and kinds of AA, as well as their sequence in the polypeptide ✓ GH
chain • Connective tissue structure, examples:
• Crucial for the function and molecular characteristics of the protein ✓ collagen (major fibrous element of the skin, bone, tendon, cartilage, blood
2. Secondary Structure vessels, and teeth)
• Winding of the polypeptide chain ✓ elastin
• Pleated sheet, alpha helix, and random coil • Biocatalysts (enzymes)
• Maintained by hydrogen bonds between the NH and CO groups of the • Participation in the hemostasis and coagulation of blood
peptide bonds
Plasma Proteins:
• More frequently analyzed
Beta pleated sheet • More than 500 plasma proteins have identified
Alpha helix random
1. PREALBUMIN (transthyretin) ✓ pregnancy
• Transport thyroxine and triiodothryonine 8. HAPTOGLOBIN
• Indicator of nutritional status • Alpha-2 glycoprotein
• (-) acute phase reactant • Transport free hemoglobin in the plasma to the RES where hemoglobin is
• Decreased: degraded
✓ inflammation and malignancy • Decreased:
✓ hepatic damage ✓ Hemolytic anemia
• Increased: ✓ Megaloblastic anemia
✓ Steroids ✓ Sickle cell anemia
✓ Alcoholism • Acute phase protein → infection, neoplasia, trauma, MI
✓ Chronic renal failure 9. CERULOPLASMIN
2. ALBUMIN • Copper containing alpha-2 globulin
• Highest concentration in the serum • Acute phase reactant
• Colloid osmotic pressure of the intravascular fluid → responsible for nearly 80% of • Decreaed:
this pressure ✓ Wilson’s disease (hepatolenticular degeneration)
• Decreased: ✓ Malnutrition
✓ Malnutrition ✓ Chronic hepatits
✓ Cirrhosis 10. HEMOPEXIN
✓ Viral hepatitis • Beta-1 globulin
✓ GI inflammation • Transport protein that bind free heme after hemoglobin has been catabolized to
✓ Renal diseases its component parts
• Increased: dehydration 11. TRANSFERRIN
• (-) acute phase reactant • Beta glycoprotein that functions as an iron transport protein
3. GLOBULINS • (-) acute phase reactant → decreased in inflammation
• Alpha-1, alpha-2, beta and gamma fractions • Decreased:
4. ALPHA-1 ANTITRYPSIN ✓ Liver disease
• Acute phase reactant ✓ malnutrition
• Neutralize trypsin-like enzymes that cause hydrolytic damage to structural ✓ nephrotic syndrome
proteins 12. C-REACTIVE PROTEIN
• 90% of the fraction of serum proteins • Acute phase protein (most sensitive)
• Associated with severe, degenerative, emphysematous pulmonary disease, • Involved with the immune system and plays a role in complement activation
juvenile hepatic cirrhosis • Elevated:
• Increased: ✓ Infections
✓ Inflammatory reactions ✓ Tissue damage or necrosis
✓ Pregnancy • Early marker for acute myocardial infarction
✓ Contraceptive use 13. FIBRINOGEN
• Acute phase reactants – plasma proteins that elevate in response to acute • Acute phase protein; glycoprotein
inflammation (infection, myocardial infarction, tumor growth, surgery or trauma) • One of the largest proteins in blood plasma
5. ALPHA-1 ACID GLYCOPROTEIN • Form a fibrin clot when activated by thrombin (clottable protein)
• Inactivate progesterone 14. IMMUNOGLOBULINS
• Formation of certain membranes and fibers in association with collagen • Produced by plasma cells from B lymphocyte lineage in the BM
• Acute phase reactant A. IgG
6. ALPHA-1 FETOPROTEIN • Highest concentration in adults
• Protects the fetus from immunolytic attack by its mother, modulates cell growth; • Capable of crossing the placenta
transport compounds such as steroids; for development of the female • Neutralize toxins, bind antigens and activate complement
reproductive system • Increased:
• Screen fetuses: neural tube defects, Down’s syndrome ✓ Liver disease
• increased: ✓ Infections
✓ hemolytic disease of the newborn (HDN) ✓ Collagen disease
✓ presence of twin pregnancy B. IgM
• tumor marker: hepatocellular carcinoma • First Ig to be produced during the immune response (1˚ response to
7. ALPHA-2 MACROGLOBULIN antigen)
• Protease inhibitor • First Ig produced by the fetus during development
• Increased: • Increased:
✓ Diabetes ✓ Rubella
✓ Liver disease ✓ Herpes
✓ Use of contraceptive medications ✓ Syphilis
 C. IgA
• Present in the respiratory and GI mucosa, saliva, tears, sweat
• Provides external surface protection against microorganisms
D. IgE Dye Binding Method:
• Firmly bound to mast cells • Based on the ability of most proteins in serum to bind dyes
• Associated with allergic and anaphylactic reactions • Bromphenol blue, Ponceau S, Amido Black 10B, Lisamine Green, Coomassie
brilliant blue
• Simple and fast
Miscellaneous Proteins:
1. MYOGLOBIN Ultraviolet Absorption:
• Heme protein found in the striated skeletal and cardiac muscles • Protein → estimated by the use of UV spectrophotometry
• Acute myocardial infarction (AMI) → increase seen within 1-3 hours of onset, • Proteins absorb light at 280 nm and 210 nm
peak concentration in 5-12 hours • 280 nm → related to the absorbance of tyrosine, tryptophan, and phenylalanine
• Degree of elevation indicates the size of infarct • 210 nm → absorbance of the peptide bond
• Not cardiac specific
2. TROPONIN
• Consists of troponin T, I and C QUANTITATION OF SPECIFIC PROTEINS:
• Complex of proteins that bind to the filaments of striated muscles but are not Refractometry:
present in smooth muscle • Rapid, requires a very small sample size
• Troponin I → highly specific for myocardial tissue; sensitive indicator of even minor • Velocity of light is changes as it passes the boundary between two transparent
amount of cardiac necrosis layers (air and H2O), causing the light to bend (refracted)
• Troponin I → increase 3-6 hours of onset; peak 14-20 hours • Refractive index of sample is measured in a refractometer
• Troponin T → rise within 3-4 hours; peak 10-24 hours • A drop of serum is placed by capillary action between coverglass and prism
• The refractometer is held so light is refracted thru serum layer
• The refracted rays cause part of the field of view to be light, producing a point at
QUANTITATION OF SERUM TOTAL PROTEINS: which there is a sharp line between light and dark
Kjeldah Method: • Majority of solids dissolved in serum are CHON; refractive index reflects
• Based on the measurement of nitrogen concentration of CHON
• Serum proteins are precipitated with organic acid (TCA or tungstic acid) • Disadvantage: other substances (electrolytes, glucose, urea) contributes to
• Npn (nonprotein nitrogen) is removed with the supernatant refractive index
• Protein → digested in H2SO4 with heat (340-360˚C) and a catalyst such as cupric • Lipemia, hemolysis, bilirubin, temperature dependent
sulfate, to speed the reaction
• Potassium sulfate → to increase the boiling point to improve the efficiency of Salt Fractionation:
digestion • Globulin can be separated from albumin by salting out using sodium salts
• H2SO4 → oxidizes the C, H, and S in protein to CO2, CO, H2O and SO2 • Salts by decreasing the water available for hydration of hydrophilic groups will
• Nitrogen in the protein is converted to ammonium bisulfate (NH4HSO4) → then cause precipitation of the globulins
measured by adding alkali and distilling the ammonia into a standard boric acid • 26-28% (w/v) Na2SO4, Na 2SO3
solution
• Ammonium borate (NH4H2BO3) → titrated with HCl to determine the amount of Dye Binding:
nitrogen • Most widely used
• Disadvantage: time consuming; too tedious for routine use • pH of the solution is adjusted so that albumin is positively charged
• albumin is attracted to and bind to an anionic dye
Biuret Method: • methyl orange → non specific for albumin
• Most widely used method • HABA (2,4 hydroxyazobenzene benzoic acid) → more specific for albumin;
• Cupric ions complex with the groups involved in the peptide bond salicylates, penicillin, bilirubin interfere with the binding
• In alkali medium, and in the presence of at least 2 peptide bonds → a violet- • BCG (bromcresol green) → not affected by interfering substances (bilirubin,
colored chelate is formed salicylates); hemoglobin can bind to the dye; alpha globulins would react with
• Sodium potassium tartrate → to complex cupric ions to prevent their precipitation BCG
in the alkali solution • BCP (bromcresol purple) → not subjected to most interferences; in patients with
• Measured at 540 nm renal insufficiency, BCP underestimate the serum albumin
• The color that is formed is proportional to the number of peptide bonds present
and reflects the total protein level
• Reagents: DETERMINATION OF TOTAL GLOBULINS:
➢ Copper sulfate as major reactant • By direct colorimetric method using glyoxylic acid
➢ NaOH to establish an alkali environment • Glyoxylic acid, in the presence of Cu+2 and in acid medium (acetic acid and
➢ Stabilizers (eg potassium iodide) to keep copper in the cupric (Cu+2) state H2SO4) condenses with tryptophan found in globulins to produce a purple color
• Albumin can be calculated by subtraction of the globulin from total protein
 TOTAL PROTEIN ABNORMALITIES:
Hypoproteinemia:
• (-) nitrogen balance
• Excessive loss of plasma proteins
• Ex. nephrotic syndrome, leakage of GIT in inflammation, loss of blood in open
wounds, internal bleeding or extensive burns
• Decrease intake → malnutrition, malabsorption
• Decrease synthesis → liver disease

Hyperproteinemia:
• An increase in total plasma proteins
• Not seen as commonly as hypoproteinemia
• Dehydration → vomiting, diarrhea, excessive sweating, diabetic acidosis,
hypoaldosteronism
• May be a result of excessive production, primarily of the gamma-globulins
(multiple myeloma, macroglobulinemia)

➢ Reference interval for serum TP:

≈ 6.5 – 8.3 g/dL (65-83 g/L) for ambulatory adults
≈ 6.0 – 7.8 g/dL (60-78 g/L) in recumbent position (a result of shifts in water
distribution in the extracellular compartments)

➢ Serum albumin concentration:

≈ 3.4 – 5.0 g/dL (34-50 g/L)