Protein Characteristics and Structures

Characteristic of Proteins

• Proteins are macromolecules.
• Proteins are made of amino acids linked by peptide bonds.
• Peptide bonds form when the carboxyl group (-COOH) of one amino acid links to the amino group (-NH2) of another, releasing water.
• N-terminal is the end of a protein structure with a free amino group.
• C-terminal is the end of a protein structure with a free carboxyl group.
• Proteins contain 16% nitrogen which distinguishes them from carbohydrates and lipids.

Protein Structure

• Primary Structure: Amino acids are linked through covalent peptide bonds in a specific sequence, forming a polypeptide chain.
• Secondary Structure: The polypeptide chain winds into alpha-helices and beta-sheets via hydrogen bonds between CO and NH groups of peptide bonds.
• Tertiary Structure: The coiled polypeptide chain folds into a 3D structure through R group interactions like disulfide linkages, hydrogen bonds, and van der Waals forces.
• Quaternary Structure: Two or more folded polypeptide chains bind via hydrogen bonds and electrostatic interactions forming a functional protein.

Classification of Proteins

• Simple proteins are polypeptides made only of amino acids.
• Globular proteins have symmetrical, compact folded polypeptide chains; an example is albumin.
• Fibrous proteins have elongated, asymmetrical polypeptide chains; examples include troponin and collagen.
• Conjugated proteins are composed of protein (apoprotein) and nonprotein (prosthetic group) components.
• Prosthetic groups of conjugated proteins are commonly metal, lipid, and carbohydrate.
• Metalloproteins have a metal prosthetic group; an example is ceruloplasmin.
• Lipoproteins have a lipid prosthetic group; examples are cholesterol and triglyceride.
• Glycoproteins have 10-40% carbohydrates attached; haptoglobin is an example.
• Mucoproteins have >40% carbohydrates attached; an example is mucin.
• Nucleoproteins have DNA or RNA nucleic acids attached; chromatin is an example.

Protein Functions

• Energy Production: Proteins break down into amino acids used in the citric acid cycle to produce energy.
• Water Distribution: Proteins maintain colloidal osmotic pressure between body compartments.
• Buffer: The individual amino acid R groups of a protein provide buffering capacity by binding or releasing H+ ions.
• Transporter: Proteins bind and allow the movement of hormones, free hemoglobin, lipids, drugs, calcium, and unconjugated bilirubin in circulation.
• Antibodies: Proteins protect against "foreign" invaders.
• Cellular proteins can function as receptors for hormones to activate cellular components.
• Collagen is a structural protein that maintains the structure of skin, bone, cartilage, and blood vessels.
• Enzymes catalyze and accelerate chemical reactions.

Plasma Total Protein Regulation

• The liver synthesizes most plasma proteins.
• Plasma cells synthesize immunoglobulins.
• Proteins are synthesized from amino acids linked by peptide bonds.
• When proteins degrade, amino acids undergo deamination, forming ammonia, which converts to urea for excretion.
• Cytokines released at injury sites increase the liver's synthesis of acute-phase reactant proteins as a nonspecific response to inflammation, autoimmune disorders, infections, tissue damage from tumors, myocardial infarctions, trauma, or surgical procedures.
• Some proteins decrease in concentration and are negative acute-phase proteins, including prealbumin, albumin, and transferrin.
• Immunoglobulins are humoral antibodies produced to destroy foreign antigens.
• Total protein reference range: 6.5-8.3 g/dL.
• Albumin reference range: 3.5-5.0 g/dL.

Plasma Total Protein Changes

• Changes in total protein concentration can be associated with hypoproteinemia.
• Hypoproteinemia can be caused by urinary loss, gastrointestinal tract inflammation, liver disorders, malnutrition, inherited immunodeficiency disorders & extensive burns.
• Changes in total protein concentration can be associated with hyperproteinemia.
• Hyperproteinemia can be caused by dehydration and increased protein production from monoclonal and polyclonal gammopathies, and chronic inflammatory diseases.

Clinical Significance of Major Proteins

• Prealbumin (transthyretin) is an indicator of nutritional status.
• Prealbumin (transthyretin) is a protein that transports thyroid hormones.
• Prealbumin is decreased in liver disorders, inflammation, malignancy, and poor nutrition.
• Prealbumin is increased in steroid therapy, chronic renal failure, and alcoholism.
• Albumin is synthesized in the liver and is the most concentrated plasma protein.
• Albumin binds many analytes for transport in blood including bilirubin, steroids, ions, fatty acids and drugs.
• Albumin significantly contributes to plasma osmotic pressure.
• Decreased albumin can be caused by liver disorders because of decreased production, gastrointestinal diseases associated with malabsorption and muscle-wasting diseases.
• Severe burns, renal disease, starvation, and malnutrition are all possible causes of decreased albumin.
• Increased albumin can be caused by dehydration which results in relative increase.
• α1-Antitrypsin is an acute-phase reactant and protease inhibitor that neutralizes trypsin-type enzymes.
• α1-Antitrypsin deficiency can lead to emphysema-associated pulmonary disease and severe juvenile hepatic disorders.
• α1-Antitrypsin is increased in inflammatory disorders.
• α1-Fetoprotein (AFP) is made during gestation in the yolk sac and liver of the fetus.
• AFP peaks at 13 weeks and declines at 34 weeks gestation.
• Normally, the levels of AFP are very low in adults.
• Maternal serum AFP is measured between 15-20 weeks of gestation and is reported as multiples of the median (MoM).
• Increased maternal serum AFP can indicate neural tube defects, spina bifida, and fetal distress.
• Decreased maternal serum AFP can indicate Down syndrome and trisomy 18.
• Increased AFP in adults can indicate hepatocellular carcinoma and gonadal tumors.
• α1-Acid glycoprotein (orosomucoid)is an acute-phase reactant that binds to basic drugs.
• α1-Acid glycoprotein is increased in inflammatory disorders such as rheumatoid arthritis, pneumonia, and conditions associated with cell proliferation.
• α1-Acid glycoprotein is decreased in nephrotic syndrome.
• Haptoglobin is an α2-globulin that binds free hemoglobin and is an acute-phase reactant.
• Haptoglobin is increased in inflammatory conditions, burns, and trauma.
• Haptoglobin is decreased in intravascular hemolysis because of the formation of a haptoglobin-hemoglobin complex for removal by the liver.
• Ceruloplasmin is an acute-phase reactant that is an α2-globulin, copper-containing protein with enzymatic activity.
• Approximately, 90% of serum copper is bound in ceruloplasmin.
• Ceruloplasmin is increased in pregnancy, inflammatory disorders, malignancies, and intake of oral estrogen and contraceptives.
• Ceruloplasmin is decreased in Wilson disease, malnutrition, malabsorption, and severe liver disease.
• α2-Macroglobulin is a proteolytic enzyme inhibitor that inhibits thrombin, trypsin, and pepsin
• α2-Macroglobulin is increased in nephrotic syndrome, contraceptive use, pregnancy, and estrogen therapy
• α2-Macroglobulin is decreased slightly in acute inflammatory disorders and prostatic cancer.
• α2-Macroglobulin is decreased markedly in acute pancreatitis
• Transferrin: β-globulin transports iron.
• Transferrin is decreased in infections, liver disease, and nephrotic syndrome
• Transferrin is increased in iron-deficiency anemia and pregnancy.
• C-reactive protein (CRP) is a β-globulin, an acute-phase reactant.
• CRP is increased in tissue necrosis, rheumatic fever, infections, myocardial infarction, rheumatoid arthritis, and gout.
• Immunoglobulins are antibodies.
• The five major classes of Immunoglobulins are IgA, IgD, IgE, IgG, and IgM.
• Immunoglobulins are synthesized in plasma cells as an immune response.
• IgG can cross the placenta.
• IgG is increased in liver disorders, infections, and collagen disease.
• IgG is decreased in the presence of increased susceptibility to infection.
• IgA levels increase after birth.
• IgA is increased in liver disorders, infections, and autoimmune diseases.
• IgA is decreased in inhibited protein synthesis and hereditary immune disorders.
• IgM cannot cross the placenta, but is made by the fetus.
• IgM is increased in bacterial, viral, and fungal infections, and Waldenstrom macroglobulinemia.
• IgM is decreased in renal diseases with protein loss and immunodeficiency disorders.
• IgD is increased in liver disorders, infections, and connective tissue disorders.
• IgE is increased in allergies, asthma, hay fever and parasitic infections.
• Fetal fibronectin predicts risk of premature birth.
• Fibronectin is a normal constituent of the placenta and amniotic fluid.
• Increased fibronectin in cervicovaginal secretions suggests a risk for premature birth, which is caused by stress, infection, or hemorrhage.

Methodology for Serum Total Protein, Albumin and Protein Fractionation

• Refractometry measures the change in light velocity (light bending) as light passes through the boundary between air and water. -This is proportional to the concentration of solutes (proteins) in the water (serum).
• The biuret method is based on cupric ions complexing with peptide bonds in alkaline medium, to produce a purple complex. -The amount of purple complex is directly proportional to the number of peptide bonds present and reflects protein concentration.
• Dye binding techniques allow protein to bind to a dye, forming a protein dye complex resulting in absorbance shift in the dye.
• The absorbance increase is directly proportional to the protein concentration.
• Kjeldahl method: Too cumbersome for routine testing. -Best used to validate materials in the biuret method & is considered the reference method of choice. -Based on quantification of the nitrogen content of protein.
• Serum protein electrophoresis: Involves applying serum at the cathode region of an agarose gel/ cellulose acetate plate saturated with a pH 8.6 buffer. -Net negative charge serum proteins migrate toward the anode. -Albumin travels the farthest, followed by α1, α2, β, and γ globulins. -Proteins are fixed, stained, and quantified via a densitometer.
• High-resolution protein electrophoresis: Uses agarose gel, higher voltage, cooling system, and a more concentrated buffer to separate proteins into at least twelve zones.
• Isoelectric focusing is a type of zone electrophoresis where protein separation is based on the isoelectric point (pI) of proteins.
• Immunochemical methods: include homogeneous and heterogeneous immunoassays, immunonephelometry, immunoelectrophoresis, radial immunodiffusion (RID), electroimmunodiffusion, and immunofixation.

Proteins in Other Body Fluids

• Urinary proteins quantification is performed on 24-hour urine specimens using sulfosalicylic acid, trichloroacetic acid, benzethonium chloride and Coomassie brilliant blue
• Reference range (urine total protein): 1-14 mg/dL; <100 mg/day.
• Increased protein in urine can result from tubular or glomerular dysfunction, multiple myeloma, Waldenstrom macroglobulinemia, and nephrotic syndrome.
• Urinary proteins clinical significance also may include Bence Jones protein for patients with multiple myeloma and/or damaged glomerular membranes in diabetes, amyloidosis and collagen diseases.
• Glomerular dysfunction can be detected in its early stages by measuring microalbumin in urine.
• Microalbuminuria is a condition where urine albumin is greater than normal, yet cannot be detected by the urine dipstick method.
• Diabetic microalbuminuria precedes nephropathy.
• Enzyme immunoassays and immunonephelometric assays are methods for microalbumin quantification.
• The reference range for urine albumin: <30mg/day.
• CSF is an ultrafiltrate of plasma that forms in the ventricles of the brain.
• Test methods for CSF proteins include sulfosalicylic acid, trichloroacetic acid, benzethonium chloride and Coomassie brilliant blue.
• Reference range: 15-45 mg/dL.
• Viral, bacterial, fungal meningitis, traumatic tap, multiple sclerosis, herniated disk, and cerebral infarction can cause an increase in clinical significance.
• Hyperthyroidism and central nervous system leakage of CSF can cause a decrease in clinical significance.