Effect of protease binding by .alpha.2-macroglobulin on intrinsic fluorescence

Publisher Website
Google Scholar
Abstract
Intrinsic protein fluorescence as a method for investigating the reactions of [human plasma] .alpha.2-macroglobulin (.alpha.2M) with proteases and amines. Changes in fluorescence intensity of .alpha.2M in the presence of proteases and amines were correlated with structural and functional changes in the .alpha.2M molecule. Intrinsic fluorescence showed 2 mol of trypsin bound to 1 mol of .alpha.2M whereas thrombin and plasmin each bound in a stoichiometry closer to 1:1. Changes in fluorescence caused by ammonium ion parallel the loss of the ability of .alpha.2M to protect trypsin from soybean trypsin inhibitor. The exposure of SH groups on .alpha.2M by a small organic amine (methylamine) also correlated with fluorescence change that could be quantitatively eliminated by prior reaction of .alpha.2M with trypsin. Cleavage of .alpha.2M by 4 serine proteases (plasmin, thrombin, trypsin, and elastase) as determined by sodium dodecyl sulfate gel electrophoretic analyses and the binding of plasmin and thrombin as measured by macromolecular inhibitor assays corresponded to the increase in fluorescence intensity. In addition, the rate of thrombin inhibition for clotting fibrinogen was the same as the rate of fluorescence change observed when thrombin was incubated with .alpha.2M. Intrinsic protein fluorescence is an easy and rapid technique for assessing both qualitative and quantitative aspects of protease-.alpha.2M interactions.