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Studies on Human Antibodies

IX. Interaction of 1-(m-Nitrophenyl)-Flavazoles of Isomaltose Oligosaccharides with Purified Antidextrans: Quantitative Hapten-Inhibition and Fluorescence Quenching Studies^1,2,

From the Departments of Microbiology, Neurology, and Human Genetics and Development, College of Physicians and Surgeons, Columbia University, and the Neurological Institute, Presbyterian Hospital, New York 10032

Abstract

The 1-(m-nitrophenyl) flavazoles (NPF1) of the isomaltose oligosaccharides obtained by condensation of o-phenylenediamine, m-nitrophenyl hydrazine sulfate with the isomaltose oligosaccharides were used to study their interaction with human antidextran by the quantitative hapten inhibition and fluorescence-quenching techniques. The NPF1 haptens inhibited in the dextran-antidextran system in whole serum in the same order as did the isomaltose oligosaccharides; that is, NPF1 of isomaltoheptaose (NPF1IM7) was the best inhibitor, inhibitory potency decreased progressively to the NPF1 of isomaltotetraose (NPF1IM4). Studies with antidextrans purified on a specific immunoadsorbent and eluted sequentially by isomaltotriose (IM3) and isomaltohexaose (IM6) showed that NPF1IM4 and NPF1IM5 inhibited the IM3 antidextran eluate better than the IM6 eluate, confirming the previous finding that antidextrans consist of subpopulations with different size combining sites.

All NPF1 of isomaltose oligosaccharides caused specific fluorescence quenching of the purified antidextrans which could be reversed specifically by addition of unlabeled IM6. Since relatively large amounts of nonspecific attenuation also occurred with globulin devoid of antidextran activity or with L-tryptophan solution, absolute values of maximum quenching (Q_max) could not be obtained, but the relative value of Q_max for each NPF1 was estimated from Scatchard plots by using various values of Q_max and taking the one which gave an extrapolated value of two combining sites since purified antidextrans are IgG and have two combining sites/molecule. NPF1IM3 with the NPF1 group closest to the immunodominant non-reducing end of IM3, which would be closest to the antibody-combining site, gave the largest Q_max and NPF1IM7 with the NPF1 group farthest from the immunodominant end gave the smallest Q_max. Differences in Q_max values were seen for the various antidextran fractions eluted by methyl -D-glucoside, IM3 and IM6. Average intrinsic association constants, K₀, of the purified antidextrans obtained from the Scatchard plots ranged from 5 to 8 x 10⁴ for NPF1IM7 and from 1 to 1.3 x 10⁴ for NPF1IM3. NPF1IM3 and NPF1IM4 gave smaller K₀ values with the IM6 antidextran eluate than with the IM3 and methyl -D-glucoside eluates, providing further evidence that small haptens are bound less effectively in combining sites of larger size.

Footnotes

¹ Aided by grants from the National Science Foundation (GB-8341 and GB-25686) and a General Research Support grant of the United States Public Health Service to Columbia University.

² From Part III of a dissertation submitted by V. Harisdangkul in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Faculty of Pure Science, Columbia University, New York.

³ Rockefeller Foundation Fellow, 1967–1971.