Studies on proteases and α-macroglobulin activity in amphibian blood plasma

Frog plasma effectively hydrolysed the synthetic chromogenic substrates,H-D-Glu-Gly-Arg-p-nitroanilide (S-2444), benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S-2222) and acetyl-Ile-Glu-Gly-Arg- p-nitroanilide (S-2423), all sensitive substrates for trypsin. Moderate hydrolytic activities was observed with H-D-Phe-Pip-Arg-p-nitroanilide (S-2238, substrate for thrombin) and H-D-Pro-Phe-Arg-p-nitroanilide (S-2302, substrate for plasma kallikrein). Frog plasma contained moderate a-macroglobulin activity. When plasma was incubated at 37°C, the macroglobulin activity decreased in a time dependent manner while only a moderate decrease in the protease activity was observed. Ten fold dilution of plasma with 0.1 M phosphate buffer, pH 7.6 prevented the inherent loss of macroglobulin activity but it had no effect on protease activity. Dye ligand chromatography of the plasma on red Sepharose revealed that bulk of α-macroglobulin activity along with minor proteolytic activity (S-2222 hydrolysis) was present in the washings. On the other hand, about one third of the α-macroglobulin activity and bulk of the protease activity was bound to the column and were eluted with 1.5 M NaCl. α-Macroglobulin activity in red Sepharose washings and elutions on chromatography on Sephadex G-200,was eluted in two regions with Ve/Vo value of 1.33 and 1.08, respectively.