Separation and quantitation of human plasma acid stable trypsin inhibitors by RP-HPLC

Summary Human plasma contains acid stable trypsin inhibitors (ASTIs) which are present in small quantities and are bound tightly but reversibly to the enzyme. For the analysis of such endogenous inhibitors a procedure has been proposed analogous to that described by us for urinary trypsin inhibitors (UTIs). This chromatographic procedure allows a direct measurement of such substances and avoids the remarkable losses which, as a rule, are connected with the isolation methods so far used. Two ASTIs are selectively isolated by affinity chromatography on immobilized trypsin and separated by RP-HPLC when an acidified plasma sample (1 ml) is treated. These ASTIs have apparent m.w. of ca. 72000 and 18000 daltons, respectively. A third ASTI, having apparent m.w. of ca. 6000 daltons, is evidenced when at least 20 ml of plasma sample were processed with an unusual procedure which avoids the adsorption on the protein precipitate formed in acid treatment. For each ASTI determination, reproducibility, linearity range, recovery (95%) and detection limit are reported.

---

Trennung und quantitative Bestimmung von säurebeständigen Trypsininhibitoren des menschlichen Plasmas durch RP-HPLC