Immunologically Active and Structurally Similar Fragments of Protein A from Staphylococcus aureus

Abstract

To study the active site(s) in protein A, partial tryptic digestions of the protein and of intact Staphylococcus aureus were performed. Fragments which bind to the Fc‐part of human IgG were isolated by affinity chromatography on IgG‐Sepharose 4B and purified by ion‐exchange chromatography on phosphocellulose. From a partial tryptic digest of pure protein A at 30°C, pH 8.2 for 30 min we have isolated and characterized six active fragments with molecular weights ranging from 6000 to 8000. Two active fragments, obtained in high yields by digestion at pH 7.2 of intact protein‐A‐containing bacteria, were shown to be similar to two of the six characterized fragments from the digest of pure protein A. All fragments appeared to have similar amino acid sequences, judged by peptide mapping, specific staining and amino acid analysis; some are very possibly overlapping peptides. Each fragment probably contains only one active site region since all are monovalent in the Fc‐reaction when studied with a hemagglutination technique. The maximal molar yield of active fragments obtained from the digestion of pure protein A accounts for about 210% of the amount of protein A used. Thus protein A, suggested to consist of repeating units, should exhibit at least three similar if not identical active regions.

Repetitive sequences in protein a from Staphyloccus aureus: Three highly homologous Fc‐binding regions

“…wt. determinations by gel chromatography and gel electrophoresis performed on the phosphocellulose material ranged from 5500-7500 [9]. The N-terminal analyses, the homogeneity in polyacrylamide gel electrophoresis on 11% gels at pH 8.9 [9], the homogeneity in both cation and anion exchange chromatography, and the amino acid analysis data all indicate that pure fragments were obtained.…”

“…The fragment A chromatogram was identical to that of fragment II, and fragment B gave the same chromatogram as fragment IV. Because of this and the data presented in [9], fragments A and II and fragments B and IV respectively were considered to be identical products, hence fragments A and B are omitted in the following characterizations. Fragment VI was split into three compounds VI', VI" and VI"'.…”

“…1 and 2 illustrate the elution profile from the phosphocellulose column separations of Fc-binding material generated by partial tryptic digestions of isolated protein A and of intact bacteria respectively [9] . Chromatography on DEAE-cellulose columns showed that fragment A, II, VI and VII could be further separated while fragment B, I. III, IV and V gave single peaks.…”

“…wts. around 7000 were prepared as described in [9]. The isolation by chromatography on IgG-Sepharose and phosphocellulose columns were performed essentially as described in [9].…”

“…Furthermore, it was suggested that these regions were similar with respect to the amino acid sequences. Digestion of intact bacteria could also be used as a preparative method for fragments similar to those obtained by digestion of isolated protein [9].…”