Freedman H J, Wilkens H J, Back N
Res Commun Chem Pathol Pharmacol. 1976 Nov;15(3):581-99.
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.
血浆激肽原，一种长期以来被认为难以鉴定其特性的缓激肽不稳定前体，已通过加速、非破坏性方法进行了测定和部分纯化。尽管肝素在使激肽原过早消耗最小化方面比溴化六甲双铵效果更好，但两者联合使用时均无效。研究了不同孵育参数对胰蛋白酶消耗激肽原的影响。由于胰蛋白酶（50微克/毫升）在45℃下于0.1M氯化钙中10分钟释放的缓激肽与在37℃下30分钟释放的一样多，因此采用前一种条件，根据大鼠子宫生物测定法测定释放的缓激肽当量来测定激肽原。从血浆中对激肽原进行常规10倍纯化的最快（2天）且最简单的方法包括硫酸铵沉淀（33 - 46%），然后单次通过伴刀豆球蛋白A - 琼脂糖柱。伴刀豆球蛋白A比其他蛋白质（白蛋白、γ - 球蛋白）更牢固地结合糖蛋白（如激肽原）。结合时对胰激肽释放酶攻击有抗性的激肽原用0.05Mα - 甲基甘露糖苷解吸。
