The covalent-binding reaction of complement component C3

Publisher Website
Google Scholar
Abstract
The complement protein C3 [complement component 3], when activated by limited proteolysis, forms a short-lived reactive intermediate fragment, nascent C3b, which binds covalently to certain surfaces. The characteristics of the covalent binding reaction were studied by using Sepharose-trypsin as a combined proteolytic activator and binding surface for [human] C3. Binding of C3 to Sepharose-trypsin is saturable, with a maximum of 25-26 molecules of C3b bound per molecule of trypsin. A minimum life-time of about 60 .mu.s for the reactive intermediate was calculated from binding of C3 at saturation. Initial binding efficiencies of over 30% can be obtained at physiological pH and ionic strength. The efficiency of C3 binding to Sepharose-trypsin decreases as pH increases and also shows a slight decline at high ionic strength. The covalent binding of C3 to Sepharose-trypsin can be inhibited by a range of O and N nucleophiles. C3 activation in the presence of radioactive forms of 4 such nucleophiles (phenylhydrazine, methylamine, glycerol and glucosamine) results in apparent covalent nucleophile incorporation into the C3d fragment of C3. The quantity of radioactive nucleophile bound can be predicted from the observed potency of the nucleophile as an inhibitor of C3 binding to Sepharose-trypsin. The radioactive nucleophiles may be considered as active-site labels for C3.