Accelerated assay, preservation and purification (concanavalin A) of plasma kininogen.
Plasma kininogen, a labile precursor of bradykinin long considered difficult to characterize, has been assayed and partially purified by accelerated, non-destructive methods. Although heparin minimized premature kininogen consumption better than hexadimethrine bromide, both were ineffective when used in combination. The effects of varying incubation parameters upon kininogen consumption by trypsin were studied. Since trypsin (50 mug/ml) liberated in 10 min at 45degreesC in 0.1 M CaC12 as much bradykinin as in 30 min at 37 degrees C, the former conditions were adopted for assaying kininogen in terms of bradykinin equivalents released, as determined by rat uterus bioassay. The fastest (2-day) and simplest procedure for a routine 10-fold purification of kininogen from plasma consisted of ammonium sulfate precipitation (33-46%) followed by a single passage through a concanavalin A-agarose column. Con A binds glycoproteins (e.g. kininogen) more firmly than other proteins (albumin, gamma-globulins). Kininogen, resistant to killikrein attack while bound, was desorbed with 0.05 M alpha-methyl mannoside.
D001681:Biological Assay; D001920:Bradykinin / Q000097:blood; D002118:Calcium; D002846:Chromatography, Affinity; D002951:Citrates; D003208:Concanavalin A; D006493:Heparin; D006583:Hexadimethrine Bromide; D006801:Humans; D007704:Kininogens / Q000097:blood* / Q000302:isolation & purification; D008722:Methods; D011309:Preservation, Biological; D013696:Temperature; D013997:Time Factors; D014357:Trypsin; D014361:Trypsin Inhibitors
D002951:Citrates; D007704:Kininogens; D014361:Trypsin Inhibitors; D003208:Concanavalin A; D006583:Hexadimethrine Bromide; D006493:Heparin; D014357:Trypsin; D001920:Bradykinin; D002118:Calcium