Effect of protease binding by alpha 2-macroglobulin on intrinsic fluorescence.
We have evaluated intrinsic protein fluorescence as a method for investigating the reactions of alpha 2-macroglobulin (alpha 2M) with proteases and amines. Changes in fluorescence intensity of alpha 2M in the presence of proteases and amines were shown to correlate with structural and functional changes in the alpha 2M molecule. By intrinsic fluorescence we found that 2 mol of trypsin bound to 1 mol of alpha 2M whereas thrombin and plasmin each bound in a stoichiometry closer to 1:1. Studies showed that changes in fluorescence caused by ammonium ion paralleled the loss of the ability of alpha 2M to protect trypsin from soybean trypsin inhibitor. The exposure of sulfhydryl 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 four 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. Our results indicate that intrinsic protein fluorescence is an easy and rapid technique for assessing both qualitative and quantitative aspects of protease-alpha 2M interactions.
D000641:Ammonia / Q000494:pharmacology; D001665:Binding Sites; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008744:Methylamines / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000302:isolation & purification / Q000378:metabolism*
D008744:Methylamines; D000511:alpha-Macroglobulins; D000641:Ammonia; C027451:methylamine; D010447:Peptide Hydrolases; D014357:Trypsin; D013917:Thrombin; D005341:Fibrinolysin