Assay of trypsin activity of human serum with a chromogenic substrate, CBZ-diglycyl-l-arginyl-2-naphthylamide hydrochloride (GGANA)

Recent development of a very sensitive chromogenic substrate (GGANA) for trypsin and reports of proteins (P) in serum which form inhibitor-resistant active complexes (PT) with trypsin (T) have prompted an investigation of trypsin-like activity of serum. GGANA was sensitive to T and to normal human serum but was not affected noticeably by proteolytic enzymes of serum once considered trypsin-like (that is, thrombin, fibrinolysin, and kallikrein). Though serum inhibited T to some degree, GGANA could be used to measure small quantities of T added to serum (as little as 0.05 μg./ml.). The rate of inhibition of the tryptic activity, however, was inversely proportional to the concentration of the T in the serum. Presumably this phenomenon occurs because the fraction of T that can be bound by the small quantity of high molecular weight protective protein (P) to form the active complex PT in the presence of inhibitors (I) is closer to 100% in a large excess of serum which can provide an effective concentration of P. In the absence of an effective concentration of P some of the T can be bound by I to form inactive IT, PT is irreversibly inactivated by acid while IT apparently gains some activity on acid treatment followed by neutralization. Since normal serum irreversibly loses its activity on acid treatment, it probably contains PT (endogenous). How PT, P, I, and IT activity of serum changes with acute pancreatitis would be difficult to predict. The serum trypsin-like activity of 171 normal individuals averaged 391 Klett units per 30 minutes with an upper limit of 785 and a lower limit of -4. The activity found for four cases of acute pancreatitis averaged -19. The completed study of the variation of PT in serum in pancreatitis using the chromogenic peptide, GGANA, as a substrate will be reported later.