Preparation and initial characterization of an intermediate, half-cleaved form of human .alpha.2-macroglobulin

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Abstract
A form of human .alpha.2-macroglobulin (.alpha.2M) has been prepared that has properties intermediate to those of native .alpha.2-macroglobulin and 2:1 protease-.alpha.2M ternary complex by using Sepharose-linked chymotrypsin. The intermediate form has mobility on native polyacrylamide gels between the fast and slow forms of .alpha.2M and migrates as a diffuse band. Two bait regions and two thiol esters per .alpha.2M tetramer are cleaved, although no chymotrypsin is detectable in the modified .alpha.2-macroglobulin species. The remaining bait regions and thiol esters can be cleaved by further reaction with other proteases. Intermediate-form .alpha.2M can trap 1.18 mol of chymotrypsin, 0.85 mol of trypsin, and 0.65 mol of thrombin. Although both thrombin and methylamine react with intermediate-form .alpha.2M at rates not distinguishable within experimental error from those of their reactions with native .alpha.2M, chymotrypsin-Sepharose reacts much more slowly with the intermediate form than with native .alpha.2M, indicating a nonequivalence of the two reactive sites on .alpha.2M. This nonequivalance may be present initially or be induced by reaction at the first site. Comparison of ESR results obtained from spin-labeling methylamine-treated or protease-reacted .alpha.2M with those from spin-labeling of the free SH groups in intermediate-form .alpha.2M shows that trapped protease influences the mobility of the attached nitroxide either through direct contact or by producing a different conformation from that present in methylamine-treated or intermediate-form .alpha.2M.