Clinical Chemistry Lab - 06 Protein Methodologies

CLINICAL CHEMISTRY LABORATORY PROTEIN METHODOLOGIES BIURET METHOD • • TOTAL PROTEIN • • • • • • • Protein (macromolecules with CHON) is subdivided into two o Albumin and globulin group1 Total protein = albumin + globulin Specimen: serum (not plasma) o Serum lack fibrinogen (within β-globulin group; which can falsely elevate total protein concentration) Fasting is not required o Diet has minimal effect in total protein Interferences: lipemia, hemolysis o Lipemia - turbidity of serum due to increased TAG/CM ▪ VLDL are lipoproteins which can be measured ▪ Can cause false increase o Hemolysis - bursting/lysis of RBCs ▪ Liberation of Hgb (composed of heme & globin) ▪ Globin is a protein which can cause false increase Reference value: 6.5-8.3 g/dL Reference value (supine): 6.0-7.8 g/dL o Due to fluid shift and serum albumin reduction ▪ Fluid shift - fluid in extravascular space move into intravascular space during prolonged supine causing dilution in blood vessels o Supine patients - admitted/bed-ridden patients • • • • FOLIN-CIOCALTEU (LOWRY) METHODo • KJELDAHL METHOD • • • • • • Classical method for protein quantitation Reference method but not routinely used o Reasons why not routinely used ▪ Process is time-consuming ▪ Only measures of nitrogen in specimen Measures the amount of nitrogen in specimen Assumes that nitrogen content of protein is 16% o Nitrogen content of protein varies from 15.1-16.8% 1 g of nitrogen = 6.54 g of protein Most widely used method for total protein determination A colorimetric non-enzymatic method o End product has color o Uses biuret reagent instead of enzyme ▪ Contain cupric ions which have color Absorbance of color is read at 540 nm (spectrophotometry) The absorbance of the color is proportional to the number of peptide bonds present and reflects the total protein concentration of the specimen o Peptide bonds - link amino acids o Requires at least 2 peptide bonds to have a reaction Interference: Lipemia Principle: Peptide bonds present in protein forms a bond with cupric ions forming a violet-colored chelate. • • Has the highest analytical sensitivity o Can measure very small amount of protein Principle: Oxidation of phenolic compounds such as tyrosine, tryptophan, and histidine to give a deep blue color. o Tyr, Trp, His - aromatic amino acids Main reagent: Phosphotungstic-molybdic acid or phenol reagent (oxidize phenolic compounds producing blue color) Color enhancer: Biuret reagent o Final color is measured at 750 nm 3 STEPS • • • Digestion o Decomposition of protein sample utilizing concentrated acid solution (H2SO4), a catalyst (CuSO4), and boiling point increasing agent (K2SO4) to produce Ammonium sulfate Distillation o Conversion of NH4 to ammonium bisulfite, which is then measured by adding alkali and distilling the ammonia into a standard boric acid solution forming ammonium borate Titration o The amount of ammonia (NH4H2BO3) is measured by titration using hydrochloric acid UV ABSORPTION METHOD • • • Principle: The absorbance of proteins at 210nm is due to the absorbance of the peptide bonds at specific wavelength. Proteins absorb light at 280nm and 210nm Absorption @ 280nm: tryptophan, tyrosine, & phenylalanine. o All with aromatic rings OTHER METHODS • • • Refractometry – based on measurement of refractive index of serum total proteins Turbidimetric and Nephelometric Methods o Measurement depends on formation of a uniform fine precipitate which scatters incident light in suspension (nephelometry) or block light (turbidimetry) Coomassie Brilliant Blue Dye – used for detection of proteins as little as 1μg • Ninhydrin Test – develops violet color (end color) by reacting with primary amines widely used for detection of peptides and amino acids after paper chromatography o Do not react when amine group is not a primary amine/connected to central carbon o Proline - only amino acids with no primary amino group; a imino group ALBUMIN • • • • Most widely used method for albumin are dye binding procedures. o Colorimetric non-enzymatic methods The pH of the solution is adjusted so that albumin is positively charged and binds to an anionic dye (- charged) o Proteins are amphoteric (+/- charge) ▪ Net charge depends on the pH they are dissolved The concentration of albumin is calculated by measuring the absorbance of the albumin-dye complex which is proportional to the specimen’s albumin concentration. Dyes used for measurement: o Bromcresol Green (BCG) – most commonly used o Bromcresol Purple (BCP) – most specific dye o Methyl Orange – not specific for albumin; can bind other proteins o 2,4’-hydroxyazobenzene-benzoic acid (HABA) – more specific to albumin than methyl orange but conjugated bilirubin, aspirin, penicillin, sulfonamides may interfere with the dye binding GLOBULIN • Globulin = Total Protein - Albumin