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Immunologic Alterations in Bovine Serum Albumin Resulting from Partial or Complete Reduction and Alkylation¹

Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115

Abstract

Purified monomer (4S) and dimer (7S) bovine serum albumin (BSA) were isolated from solutions of crystalline BSA by gel filtration. Mercaptoethanol reduction of BSA in buffered saline resulted in the appearance of only three sulfhydryl groups per molecule (3-SH BSA). Complete reduction in 8 M urea produced 34 free sulfhydryl groups per molecule (34-SH BSA). These BSA preparations were studied by optical rotatory dispersion, ultracentrifugation, quantitative precipitation and hemagglutination with homologous and heterologous antisera, and immunologic cutaneous reactions.

Significant distortion of the native BSA conformation by complete reduction and carboxymethylation (34-SH BSA) resulted in loss of precipitation with anti-native BSA. Partial unfolding of the native conformation of BSA resulted in greater precipitation with anti-native BSA and greater reactivity with anti-3-SH BSA hemagglutinins. This increase in antigenic reactivity was directly proportional to the loss of native -helix content and accordingly was greater for 3-SH BSA than dimer native BSA. The augmented antigenic binding is felt to be due to an exposure of hidden antigenic determinants or the creation of new determinants associated with these conformational alterations.

Completely reduced carboxymethylated 34-SH BSA is a potent immunogen inducing high titers of non-IgG antibodies with passive hemagglutination and cutaneous anaphylactic activities, but no precipitating ability. Monomer 34-SH BSA does not precipitate or fix complement with any anti-BSA antisera, and therefore may be a univalent antigen. The strong Arthus reaction produced by the 34-SH BSA immunologic system despite a lack of precipitation and complement fixation in vitro suggests the possibility of antigenic aggregation in vivo.

Footnotes

¹ This work was supported by Grant A1 05691 from the United States Public Health Service.

² Present address for reprint requests: Department of Medicine, Robert Breck Brigham Hospital, Boston, Massachusetts 02120.

³ This work was done during the tenure of an Advanced Research Fellowship of the American Heart Association.