Antibody-enzyme analogy. Characterization of antibodies to phosphopyridoxyltyrosine derivatives

• Published in: Biochemistry (Easton) 1975-02, Vol.14 (3), p.584-591
• Main Authors: Raso, Vic, Stollar, B. D
• Format: Article
• Language: English
• Subjects: Animals; Antibodies - isolation & purification; Antigen-Antibody Reactions; Binding Sites, Antibody; Carrier Proteins; Chemical Precipitation; gamma-Globulins; Haptens; Protein Binding; Pyridoxal Phosphate - immunology; Rabbits - immunology; Schiff Bases; Structure-Activity Relationship; Tyrosine - immunology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00674a019

Stable analogs of the crucial Schiff base intermediate of enzymatic and nonenzymatic pyridoxal phosphate catalysis have been used as haptens for induction of specific antibodies. N-(5-phosphopyridoxyl)-3'-amino-L-tyrosine and its conformationally distinct cyclized derivative resemble the Schiff base formed upon mixing tyrosine with pyridoxal phosphate. These compounds were covalently coupled to a protein carrier via the 3'-amino group so as to confer a prescribed orientation, with the coenzyme region farthest removed from the carrier. A third antigen, with the phosphopyridoxyl group alone as the hapten, was prepared by linkage of pyridoxal phosphate directly to free amino groups on the carrier protein. Antibodies elicited for each determinant were purified by means of appropriate affinity columns. Antibody heterogeneity was observed in that different species could be separated from a given serum by sequential elution from the affinity columns with 1 M sodium phosphate buffers of pH 7.6, 5.2, 2.6 and 1.5. In assays of quantitative precipitation, inhibition of precipitation, equilibrium dialysis, and fluorescence quenching, antibodies to the phosphopyridoxyltyrosine haptens showed specificity for the phosphorylated form of the coenzyme and binding activity for both the coenzyme and tyrosine portions of the hapten. Antibodies to the phosphopyridoxyl groups alone did not display a similar reactivity toward the tyrosine portion of the complex haptens. The cyclic and noncyclic conformations of the hapten were serologically distinct, as antibody to each reacted preferentially with the homologous form.