Isolation, by partial pepsin digestion, of the three collagen-like regions present in subcomponent Clq of the first component of human complement.
1. Digestion of human subcomponent C1q with pepsin at pH4.45 for 20h at 37 degrees C fragmented most of the non-collagen-like amino acid sequences in the molecule to small peptides, whereas the entire regions of collagen-like sequence that comprised 38% by weight of the subcomponent C1q were left intact. 2. The collagen-like fraction of the digest was eluted in the void volume of a Sephadex G-200 column, was was showm to be composed of two major fragments when examined by electrophoresis on polyacrylamide gels run in buffers containing sodium dodecyl sulphate. These fragments were separated on CM-cellulose at pH4.9 in buffers containing 7.5M-urea. 3. Human subcomponent C1q on reduction and alkylation yields equimolar amounnts of three chains, which have been designated A, B and C [Reid et al. (1972) Biochem. J. 130, 749-763]. One of the pepsin fragments was shown to be composed of the N-terminal 95 residues of the A chain linked, via residue A4, by a single disulphide bond to a residue in the sequence B2-B6 in the N-terminal 91 residues of the B chain. The second pepsin fragment was shown to be composed of a disulphide-linked dimer of the N-terminal 94 residues of the C chain, the only disulphide bond being located at residue C4.4. The mol. wts. of the unoxidized and oxidized pepsin fragments were estimated from their amino acid compositions to be 20 000 and 18 200 for the A-B and C-C dimers and 11 400, 8800 and 9600 for the collagen-like fragments of the A, B and C chains respectively. Estimation of the molecular weights of the peptic fragments by polyacrylamide-gel electrophoresis run in the presence of sodium dodecyl sulphate gave values that were approx. 50% higher than expected from the amino acid sequence data. This is probably due to the high collagen-like sequence content of these fragments.
D000478:Alkylation; D000595:Amino Acid Sequence; D002268:Carboxypeptidases; D002498:Centrifugation; D002845:Chromatography; D002847:Chromatography, Agarose; D003172:Complement C1 / Q000032:analysis*; D003165:Complement System Proteins / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D003012:Microbial Collagenase; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010434:Pepsin A; D010446:Peptide Fragments / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate
D003172:Complement C1; D010446:Peptide Fragments; D012967:Sodium Dodecyl Sulfate; D003165:Complement System Proteins; D002268:Carboxypeptidases; D010434:Pepsin A; D003012:Microbial Collagenase