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The Journal of Immunology, 1961, 86: 489-495.
Copyright © 1961 by The American Association of Immunologists, Inc.
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Nature of the Active Site of Antibenzoate Antibodies: Further Evidence for the Presence of Tyrosine1,2,

David Pressman, Alfred Nisonoff, Gerald Radzimski and Anita Shaw

From the Department of Biochemistry Research, Roswell Park Memorial Institute, Buffalo, New York

Abstract

The effect of variation of pH on the binding constant of anti-p-azobenzoate antibody with hapten indicates that there is an ionizable group in its site with a pK of about 10. This pH dependence of binding is consistent with the presence of a tyrosine, cysteine, lysine, or N terminal amino acid residue in the site. However, it has already been pointed out that an amino group (lysine or N terminal acid), if in the site, would have to be very resistant to acetylation compared to the other such groups in the antibody. The fact that the binding constant is the same at pH 6 as at 8 rules out the possiblity of histidine in the site.

The complete reversibility of the capacity to bind hapten after exposure of the antibody to pH 10 followed by restoration of neutral pH indicates that major irreversible structural changes in the antibody do not occur. Moreover, the total number of combining sites estimated by extrapolation of the binding curves to infinite free hapten concentration are the same within experimental error over the range of pH used.

Further evidence for the presence of a tyrosine in the site is the fact that the decrease in binding observed with extensive acetylation is reversible on exposure to high pH values. Such reversibility is consistent with the hydrolysis of an ester linkage rather than hydrolysis of an amide linkage and indicates the presence of tyrosine, serine or threonine or perhaps cysteine. The presence of tyrosine had previously been implicated through the effect of iodination on the antibody activity.

The reversal of acetylation is not complete. It may be that some irreversible alteration takes place in the molecule upon acetylation or that certain of the antibody molecules which have been acetylated are resistant to hydrolysis. This also suggests the possibility that the combining sites are heterogeneous with respect to chemical composition.

Footnotes

1 Presented at the 44th Annual Meeting of the American Association of Immunologists in Chicago, April, 1960.

2 Supported in part by the National Institute of Allergy and Infectious Diseases, (Grant E 2342), U. S. Public Health Service.






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