Serum Trypsin Inhibitor: A Comparison of Assay Methods Using Amide and Casein Substrates

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Abstract
Summary. Less observed trypsin inhibitor activity, at times significant in amount, was noted using benzoyl-DL-arginine-p-nitroanilide (BAPA) as a trypsin substrate than with a similar method using casein, with or without calcium in the system. When increasing amounts of serum were added to a constant amount of trypsin, the observed trypsin inhibitor activity was more nearly linear with the casein method. At high serum concentrations the amount of trypsin inhibitor activity remained relatively constant using casein substrate, while with BAPA the amount of inhibitor activity showed a fall with large excesses of serum. Trypsin inhibitor analysis of serum fractions obtained by Sephadex G-100 chromatography revealed that not only was trypsin inhibitor activity less with BAPA than with casein, but, in certain fractions, more enzymatic activity was present than could be ascribed to the initial trypsin used. It is suggested that the differences observed using BAPA and casein substrates may be the result of several possible factors including: (1) formation of a trypsin-α(2)-macroglobulin complex having differential hydrolytic activity for BAPA and casein, (2) trypsin activation of esterases or proteases from precursors and (3) differential results dependent on the interplay of various substrates with enzyme after alteration of the latter by inhibitor. It would appear that more accurate results can be obtained using casein substrate than BAPA substrate in studies of serum trypsin inhibitor activity.