Establishment of a serine protease inhibitor activity determination method by ultraviolet spectrometry

Objective To establish a determination method for serine protease inhibitor activity. Methods The inhibition constants of 2 serine protease inhibitors, recombinant Textilinin-1 (Rtxln-1) and aprotinin (AP), were determined at the same time using temperature-controlled ultraviolet spectrophotometer. The linear range and incubation temperature were optimized. The method was used to determine Rtxln-1 and AP activities, and was verified in Rtxln-1 activity determination. Results The standard curves of both inhibitors had fitting coefficient of determination ≥0.98. The linear range was 10-50 nmol/L for Rtxln-1 and 0.056-0.560 nmol/L for AP. The optimal incubation temperature for Rtxlin-1 was 37 ℃, and coefficients of variation in all precision tests were ≤5%. Conclusions The established method has high specificity, optimal curve fitting, good precision for Rtxln-1 activity determination. The method can be used for assessment of activity for trypsin inhibitors such as Rtxln-1 and AP, and for determination of general serine protease inhibitor activity. Key words: Serine protease inhibitors; Trypsin inhibitors; Spectrophotometry, ultraviolet; Activity assay