Localization of the binding site for streptococcal protein G on human serum albumin. Identification of a 5.5-kilodalton protein G binding albumin fragment.
Protein G is a streptococcal cell wall protein with separate and repetitively arranged binding domains for immunoglobulin G (IgG) and human serum albumin (HSA). In this work, the binding of protein G to HSA was studied. The results suggest that a single binding site is present on HSA: the apparent size of the HSA-protein G complex (230 kDa) corresponded to two or three HSA molecules bound to one protein G molecule, and Ouchterlony immunodiffusion did not yield any precipitate between protein G and HSA. HSA was cleaved by pepsin and CNBr into several fragments which were identified by SDS-PAGE and N-terminal amino acid sequencing, and the binding of protein G to the fragments was studied in Western blot experiments. The results indicated that the binding area was located in disulfide loops 6-8, involving both the second (loop 6) and the third (loops 7 and 8) domain of HSA. One of the protein G binding pepsin fragments, with an apparent molecular mass of 5.5 kDa, located in loops 7 and 8, was isolated and found to completely inhibit the binding between protein G and the intact HSA, again suggesting a single protein G binding site on serum albumin. Reducing the disulfide bonds of HSA, and subsequent alkylation of the half-cystine residues, significantly decreased the affinity for protein G. Protein G bound to albumin from baboon, cat, guinea pig, hamster, hen, horse, man, mouse, and rat, but not to albumin from cow, dog, goat, pig, rabbit, sheep, snake, or turkey.
Falkenberg
C
C
Department of Medical and Physiological Chemistry, University of Lund, Sweden.
Björck
L
L
Akerström
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002415:Cats; D002417:Cattle; D006224:Cricetinae; D003488:Cyanogen Bromide; D004285:Dogs; D006168:Guinea Pigs; D006736:Horses; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D010215:Papio; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry*; D010449:Peptide Mapping; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D012709:Serum Albumin / Q000737:chemistry*; D012756:Sheep; D013045:Species Specificity; D013291:Streptococcus / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013552:Swine
C029152:G-substrate; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D012709:Serum Albumin; D010434:Pepsin A; D003488:Cyanogen Bromide