Circular dichroism of C3a anaphylatoxin. Effects of pH, heat, guanidinium chloride, and mercaptoethanol on conformation and function.
Circular dichroism spectra for C3a anaphylatoxins (protein fragments generated enzymatically in serum during activation of the third component of complement (C3)) from human and porcine sources were compared in the region of 190 to 250 nm. The spectra were indistinguishable in this region, although an estimated difference of approximately 20% exists between the primary structures of human and porcine C3a. Calculations indicated a total of 40 to 45% alpha helical content based on either the 208 or 222 nm extremum of the CD spectra. Addition of either mercaptoethanol or 6 M guanidinium chloride to human C3a produced a marked decrease in the mean residue ellipticity centered at 222 nm without irreversibly affecting the biological activity. Simultaneous addition of mercaptoethanol and guanidinium chloride virtually eliminated the CD contribution at 222 nm and resulted in more than 90% inactivation of the anaphylatoxin. Removal of the denaturant and reducing agent restored both the biological activity and the CD spectrum of C3a. Heat treatment or reduction and alkylation of human C3a produced biologically inactive and conformationally modified anaphylatoxin. In contrast, C3a inactivated by enzymatic removal of the COOH-terminal arginyl residue was structurally unchanged as judged by CD measurements. Consequently, it is proposed that in addition to the essential COOH-terminal arginyl residue, a highly ordered conformation is required for biological functionality of the C3a molecule.
D000818:Animals; D001665:Binding Sites; D002268:Carboxypeptidases; D002942:Circular Dichroism; D003165:Complement System Proteins / Q000378:metabolism; D004355:Drug Stability; D006146:Guanidines; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008623:Mercaptoethanol; D011485:Protein Binding; D011487:Protein Conformation; D013045:Species Specificity; D013056:Spectrophotometry, Ultraviolet; D013552:Swine; D013997:Time Factors
D006146:Guanidines; D008623:Mercaptoethanol; D003165:Complement System Proteins; D002268:Carboxypeptidases