More useful maleimide compounds for the conjugation of Fab' to horseradish peroxidase through thiol groups in the hinge.

Nine different maleimide compounds were evaluated for the conjugation of Fab' to horseradish peroxidase through thiol groups in the hinge. The compounds evaluated were succinimidyl maleimidoacetate (I), succinimidyl 4-maleimidobutyrate (II), succinimidyl 6-maleimidohexanoate (III), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (IV), succinimidyl m-maleimidobenzoate (V), succinimidyl 4-(p-maleimidophenyl)butyrate (VI), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (VII), sulfosuccinimidyl m-maleimidobenzoate (VIII), and sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (IX). Maleimide groups of I-IV and VII were fairly stable at pH 7.0 at 30 degrees C, while those of the other compounds were significantly decomposed. I-III and VII-IX were sufficiently soluble in the reaction mixture for the introduction of maleimide groups, while the others were more or less precipitated during the reaction. II and III were the most effective in the introduction of maleimide groups and gave the highest recovery of peroxidase in the conjugate, which reached 80%. From these results, II and III were judged to be the most useful, and IV and VII were judged to be fairly useful.