Centrifugal analysis for plasma kallikrein activity, with use of the chromogenic substrate S-2302.
The amidolytic activity of activated kallikrein in plasma can be measured by use of the chromogenic substrate, S-2302 (H-D-Pro-Phe-Arg-pNA). Plasma prekallikrein was activated to kallikrein by exposure to 50 mg/L dextran sulfate in acetone/water (35/65 by vol) at 0 degrees C for 15 min. The acetone slows anti-kallikrein activity and increases the kallikrein activity by 30%. The 37 degrees C reaction mixture contained 0.54 mmol of S-2302 substrate per liter of Tris buffer (pH 7.5 at 37 degrees C). We monitored the change in absorbance at 405 nm for 60 s. The specificity of the substrate for kallikrein was demonstrated by using plasma deficient in prekallikrein (Fletcher trait) diluted with pooled normal human plasma. We recommend collecting blood specimens with sodium citrate as the anticoagulant and with use of a double-syringe technique and all-plastic containers. Plasma kallikrein activity with Chromozym-PK (Bz-Pro-Phe-Arg-pNA) as substrate (y-axis) compared with S-2302 as substrate (x-axis) gave the relation: y = 0.28x + 0.82 (r = 0.94). Day-to-day analytical variation was 2.4% for a pooled plasma with a mean value of 85.9 mukat/L. The mean (and 2 SD) for 50 healthy adults was 86.4 (32.4) mukat/L.
D000328:Adult; D002498:Centrifugation; D002863:Chromogenic Compounds; D004789:Enzyme Activation; D006801:Humans; D007610:Kallikreins / Q000097:blood*; D008722:Methods; D009842:Oligopeptides; D011288:Prekallikrein / Q000378:metabolism; D012015:Reference Standards
D002863:Chromogenic Compounds; D009842:Oligopeptides; C026148:prolyl-phenylalanyl-arginine-4-nitroanilide; D011288:Prekallikrein; D007610:Kallikreins