Immunoimmobilization of α2-macroglobulin-β-trypsin complexes: A novel approach for the biochemical characterization of modulator-protease interactions
✍ Scribed by Peter C. Harpel; Melvin B. Hayes
- Publisher
- Elsevier Science
- Year
- 1980
- Tongue
- English
- Weight
- 875 KB
- Volume
- 108
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A new approach to the biochemical characterization of a,-macroglobulin-protease complexes is described based upon our observation that cY,-macroglobulin ((YAM) or oZM-/3trypsin complexes become quantitatively bound to anti-human alM antibody that is covalently coupled to an agarose gel matrix. The immunoimmobilized qM-Ptrypsin complexes hydrolyze the chromogenic tripeptide substrate, N-benzoyl-t-phenylalanylt-valyl-t-arginine-p-nitroanilide HCI and the amidolytic activity of these complexes is proportional to the concentration of bound ptrypsin. The K, and V,,, of the soluble complexes are identical to those of the solid-phase a,M-Strypsin complexes indicating that immunoimmobilization does not alter the hydrolytic capacity of the complex. Optimal pH of the assay was 8-9, at a NaCl concentration of 0.05-0.3 M NaCI. The immobilized enzyme complex was remarkably stable since it retained its amidolytic activity following repeated assays. Varying concentrations of 1311-/3-trypsin were added to plasma containing a trace amount of '251-(r,M. The cY,M-Ptrypsin complexes that formed were quantitatively recovered from the plasma by the immunoimmobilization technique as determined by radioactivity and amidolytic activity measurements. Fifty-eight percent of the fi-trypsin added to plasma was bound to (YAM as demonstrated by both the immunoimmobilization assay and by molecular sieve chromatography. Thus a novel approach has been developed for rapid isolation and concentration of cu,M-protease complexes and for the characterization of their catalytic potential.