Protein (CC1 Lec)

Proteins

Macromolecules composed of polymers covalently linked by amino acids.

Peptide bond

Stability of the structure of proteins; bond that links amino acids.

Amino acids

Building blocks of proteins; dictate the chemical properties and biological activity of proteins.

Immunogen

Proteins with high molecular weight that are considered good in eliciting an immune response.

Transamination

Process that involves the removal of nitrogen from free amino acids, ultimately producing ammonia and ketoacids.

Nitrogen balance

The balance between protein intake and excretion.

Negative Nitrogen Balance

Occurs when protein catabolism exceeds anabolism.

Positive Nitrogen Balance

Occurs when anabolism exceeds catabolism.

Primary structure

The specific sequence of amino acids in a protein.

Secondary structure

Regularly repeating structures stabilized by hydrogen bonds within the protein, such as alpha helix or beta pleated sheet.

Responsible for the strength and flexibility of proteins.

Tertiary structure

Overall conformation of the protein molecule due to the interaction of side chains.

Quaternary structure

Interaction of more than one protein molecule or subunits.

Protein denaturation

Change in the protein structure resulting in loss of functional and chemical characteristics.

Factors that promote denaturation

1. Changes in temperature (56 deg C)

2. Hydrolysis by strong acid or alkali

3. Enzymatic action

4. Exposure to urea or other substances

5. UV light

Enzymes

Proteins that catalyze chemical reactions.

Hormones

Proteins that act as chemical messengers controlling the actions of specific cells or organs.

Transport proteins

Proteins that facilitate the movement of ions, small molecules, or macromolecules across biological membranes.

Immunoglobulins (antibodies)

Proteins produced by B-cells that mediate immune response.

Structural proteins

Fibrous proteins that provide structure to cells and tissues.

Storage proteins

Proteins that serve as reserves of metal ions and amino acids.

Plasma proteins

Proteins found in the blood plasma, serving various functions such as energy source, osmotic force, and participation in coagulation.

Acid-base balance

Participation of proteins as buffers to maintain pH.

Simple proteins

Proteins composed of only amino acids, can be globular or fibrous.

Conjugated proteins

Proteins consisting of a protein and a nonprotein prosthetic group.

Albumin

A major plasma protein that serves as a reserve source of energy and a regulator of osmotic force.

Globulin

A group of plasma proteins divided into alpha, beta, and gamma globulins based on electrophoresis fractions.

Prealbumin

Indicator of nutrition; first protein to decrease when the patient is malnourished.

Albumin

Binds bilirubin, steroids, fatty acids; major contributor to oncotic pressure; major transporter in the plasma.

α1 - Antitrypsin

Acute phase reactant; increases in acute stage of inflammation; protease inhibitor.

α1 - Fetoprotein

Principal fetal protein; tumor marker for liver cancer.

α1 - Acid glycoprotein

Acute phase reactant; increased in acute phase inflammation.

α1 – Lipoprotein

Transports lipids (HDL).

α1 - Antichymotrypsin

Inhibits serine proteinases.

Inter-α-trypsin inhibitor

Inhibits serine proteinases.

Gc-globulin

Transports Vitamin D and binds actin.

Haptoglobins

Acute phase reactant; binds hemoglobin (free hemoglobin).

Ceruloplasmin

Acute phase reactant, contains copper; low ceruloplasmin level = free copper increases.

α2 - Macroglobulin

Inhibits protease; enzyme inhibitor.

Pre-β-lipoprotein

Transports lipids (VLDL triglyceride).

Transferrin

Transport iron; inversely proportional in the iron.

Hemopexin

Acute phase reactant; binds heme.

β-Lipoprotein

Transports lipids (LDL cholesterol).

β2-Microglobulin

Component of HLA molecules.

C4, C3, C1q complement

Immune response.

Fibrinogen (Factor I)

Precursor of fibrin clot.

C-reactive protein

Major acute phase reactant; motivates phagocytosis in inflammation.

Immunoglobulin G

Antibodies; secondary response; most abundant antibody in healthy human serum.

Immunoglobulin A

Antibodies in secretions; primary response; provides mucosal immunity.

Immunoglobulin M

Antibodies in early response; biggest antibody in the human serum.

Immunoglobulin E

Antibodies (reagen, allergy); lowest concentration for healthy individuals.

Immunoglobulin D

Surface antibody.

Myoglobin

Oxygen carrier in muscles; cardiac marker (AMI).

Troponin (cTn)

Cardiac marker for acute coronary syndrome.

Brain Natriuretic peptide

Neurohormones that affect body fluid homeostasis and blood pressure; marker of congestive heart failure.

Fibronectin

Cellular interaction; adherence of the placenta to the uterus.

Cross-Linked C-Telopeptides

Proteolytic fragment of collagen I; biochemical marker of bone resorption.

β-Trace Protein

Syn:Prostaglandin D synthase; marker for CSF leakage.

Cystatin C

Cysteine proteinase inhibitor; serum marker for glomerular filtration rate.

Amyloid

Fibrous protein aggregates formed from alteration of β pleated sheaths; differential diagnosis of Alzheimer.

Hypoproteinemia

Negative balance is present; excessive loss of protein.

Hepatic Damage

Cirrhosis β-γ bridging; hepatitis ↑ γ-globulins; obstructive jaundice ↑ α2.

Hyperproteinemia

Not an actual disease state but a result of underlying cause (e.g. dehydration, excessive protein intake).

Total Protein Measurement

Reference Interval:6.5-8.3 g/dL (65-83 g/L); measurement of refractive index due to solutes in serum.

Kjeldahl Method

Reference method. Assume average nitrogen content of 16%

Kjeldahlization

The conversion of nitrogen to ammonia in the process of measuring total nitrogen in proteins.

Digestion of proteins with sulfuric acid in 340C

Nitrogen Ammonia measurement H 2SO4 →NH3

Nessler's reaction

A reaction that involves the double iodide of Hg and K, used to measure the presence of ammonia.

Ammonia + Nessler’s rgt Gum ghatti (Ammonium dimercuric iodide) → yellow solution

Berthelot reaction

A reaction that involves ammonia + alkaline hypochlorite = Na nitroprusside → formation of indophenol blue.

Refractometery

To measure the refractive index of a solution, which can indicate the presence of solutes.

Biuret

A test that involves the formation of a violet-colored chelate between Cu2+ ions and peptide bonds, used to measure protein concentration.

Measured at 540nm

Composition of Biuret

Cupric ions – breaks the peptide bonds

Tartrate salt – keeps copper in solution.

Potassium iodide – stabilizes cupric ions

Dye binding

The process in which proteins bind to dyes and cause a spectral shift in the absorbance maximum of the dye.

Albumin measurement

A method used to quantify the amount of albumin in a sample, often done using the biuret reaction.

Salt precipitation

A technique that uses sodium sulfate as a precipitating agent to separate globulins from albumin.

Globulins- precipitated in high salt concentrations.

Albumins- supernatant is quantitated by biuret reaction

Methyl Orange

Nonspecific for Albumin

HABA (2,4-hydroxyphenyl benzoic acid)

Many Interferences (salicylates, bilirubin)

BCG (Bromcresol green)

Sensitive

Overestimates low albumin levels

Most commonly used dye

BCP (Bromcresol purple)

Specific, Sensitive and Precise

Electrophoresis (Albumin measurement)

Proteins separated based on electric charge densities

Give overview in relative changes in different protein fraction

Support media for Electrophoresis

Cellulose acetate/agarose gel

Albumin reference Values of Electrophoresis

53-65% (3.5-5.0 g/dL)

α1 reference Values of Electrophoresis

2.5-5% (0.1-0.3 g/dL)

α2 reference Values of Electrophoresis

7-13% (0.6-1.0 g/dL)

β reference Values of Electrophoresis

8-14% (0.7-1.1 g/dL)

γ reference Values of Electrophoresis

12-22% (0.8-1.6 g/dL)

Acute phase reactant

Proteins that increase in concentration during inflammation or infection, such as fibrinogen, haptoglobin, Ceruloplasmin, Serum amyloid, and CRP.

High resolution protein electrophoresis

A technique that uses higher voltage and more concentrated buffer and a cooling system to separate proteins into more than 5 bands.

Turbidimetric methods (SSA, TCA, benzethonium chloride)

Proteins are precipitated as particles,

turbidimetry is measured spectrophotometrically

rapid and easy to use; accurate in sensitive methods

Folin-Lowry

Initial biuret reaction; oxidation of AA (tyrosine, etc.)

residues by Folin Phenol reagent; measurement of resultant blue color.