A study of proteases and protease-inhibitor complexes in biological fluids

TLDR

The study builds on a novel method for detecting proteolytic enzyme activity in SDS‑polyacrylamide gels. The authors screened various cell lines and fluids for plasminogen activators and examined proteases bound to α2‑macroglobulin after partial degradation, using a nonionic detergent extraction followed by electrophoresis on a fibrin‑agar indicator gel to reveal activity. The study found that plasminogen activators in urine and cell culture are lower‑molecular‑weight than in plasma, that α2‑macroglobulin‑bound proteases regain activity after SDS treatment while remaining covalently linked, and that the SDS‑polyacrylamide gel method can sensitively detect and quantify small‑MW proteinases in less than 1 µl plasma, with broad applicability to serine enzymes involved in fibrinolysis, coagulation, and kinin generation.

Abstract

We have (a) screened a variety of cell lines and body fluids for plasminogen activators and (b) studied the activity of proteases bound to α2- macroglobulin after exposing the complexes to partial degradation and/or denaturing procedures to unmask proteolytic activity. The respective results show (a) that the plasminogen activators in urine and cell culture media are generally of lower molecular weight than those in plasma; and (b) that proteases bound to α2-macroglobulin recover the ability to attack macromolecular substrates after exposure to sodium dodecyl sulfate while retaining the electrophoretic mobility of the protease inhibitor complex. This indicates that the protease and inhibitor are probably linked by covalent bonds. In contrast, other complexes formed between proteases and inhibitors of lower molecular weight (such as soybean or Kunitz inhibitors) are fully dissociated by sodium dodecyl sulfate (SDS). The experiments described were based on a new procedure for detecting proteolytic enzyme activity in SDS-polyacrylamide gels. The method relies on solutions of nonionic detergents for extracting SDS, after which the electrophoretic gel is applied to an indicator gel consisting of a fibrin- agar mixture. The method is sensitive, permitting the detection of proteinases in less than 1 μl of fresh plasma, and it is effective for resolving small differences in molecular weight. The procedure can be quantitated and, with minor modifications appropriate to each particular system, it has been applied to a broad spectrum of serine enzymes and proenzymes, including some that function in the pathways of fibrinolysis, coagulation and kinin-generation. Other potential applications appear likely.