Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent

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Abstract
A heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)propionate, was synthesized. Its N-hydroxysuccinimide ester group reacts with amino groups and the 2-pyridyl disulfide structure reacts with aliphatic thiols. A new thiolation procedure for proteins is based on this reagent. The procedure involves 2 steps. First, 2-pyridyl disulfide structures are introduced into the protein by the reaction of some of its amino groups with the N-hydroxysuccinimide ester side of the reagent. The protein-bound 2-pyridyl disulfide structures are then reduced with dithiothreitol. This reaction can be carried out without concomitant reduction of native disulfide bonds. The technique was used for the introduction of thiol groups de novo into RNase, .gamma.-globulin, .alpha.-amylase and horseradish peroxidase. N-Succinimidyl 3-(2-pyridyldithio)propionate can also be used for the preparation of protein-protein conjugates. This application is based on the fact that protein-2-pyridyl disulfide derivatives (formed from the reaction of non-thiol proteins with the reagent) react with thiol-containing proteins (with native thiols or thiolated by, e.g., the method described above) via thiol-disulfide exchange to form disulfide-linked protein-protein conjugates. This conjugation technique was used for the preparation of an .alpha.-amylase-urease, a RNase-albumin and a peroxidase-rabbit anti-(human transferrin) antibody conjugate. The disulfide bridges between the protein molecules can easily be split by reduction or by thiol-disulfide exchange. Thus conjugation is reversible. This was demonstrated by scission of the RNase-albumin and the .alpha.-amylase-urease conjugate into their components with dithiothreitol. N-Succinimidyl 3-(2-pyridyldithio)propionate was prepared in crystalline form, in which state (if protected against humidity) it is stable on storage at room temperature (23.degree. C).