The soluble dimeric beta-galactoside-binding lectin (subunit molecular mass, approximately 14 kDa) of bovine heart muscle, in common with the 14-kDa lectins of several other animal species, displays carbohydrate-binding activity when it is in the reduced state, but the purified lectin loses this activity upon oxidation. In the present study, the presence of any post-translational modification and the mechanism of the oxidative inactivation have been investigated by analyses of the reduced and oxidized forms of the purified bovine lectin by electrospray ionization-mass spectrometry (ESI-MS) and by liquid secondary ion mass spectrometry (LSIMS) of tryptic and peptic peptides. By ESI-MS, the molecular mass of the reduced lectin is determined to be 14,654.6 +/- 0.9 Da, and that of the oxidized lectin is 14,649.3 +/- 1.1 Da. These masses correspond to the amino acid sequence of the protein with the cysteines having free sulfhydryl groups in the reduced state and forming disulfide bonds in the oxidized state. There is no evidence of post-translational modification in either lectin form except for monoacetylation already predicted for alanine at the blocked N-terminal end. Pronounced differences in charge distribution in the electrospray ionization mass spectra of the reduced and oxidized lectin, reflecting a change in the number of accessible protonation sites in the oxidized protein, are consistent with the protein being held in an altered conformation by covalent bonding. The results of LSIMS analyses of tryptic and peptic peptides in conjunction with Edman sequencing indicate that disulfide bonding occurs predominantly between Cys2 and Cys130, Cys16 and Cys88, and Cys42 and Cys60. There is no evidence of oxidation of Trp68. These results, taken together with observations that almost the complete polypeptide chain is necessary for the functional integrity of the carbohydrate recognition domain (Abbott, W. M., and Feizi, T. (1991) J. Biol. Chem. 266, 5552-5557) point to intramolecular disulfide bonding with a change in protein folding and conformation as the mechanism of oxidative inactivation of the purified bovine lectin.