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Nucleic Acids Research, 1995, Vol. 23, No. 11 2037-2040
© 1995

CHEMISTRY

Maleimide-mediated protein conjugates of a nucleoside triphosphate gamma-S and an internucleotide phosphorothioate diester

Aftab S. Karim, Christian S. Johansson and Joel K. Weltman*

Division of Biology and Medicine, Brown University Providence, Rl 02912, USA

* To whom correspondence should be addressed at: 300 Hanover Street, Fall River, MA 02720, USA

Received January 9, 1995. Revised April 19, 1995. Accepted April 19, 1995.

The purpose of this study was to determine whether the gamma-S of nucleoside thlotriphosphates and the non-bridging sulfur of Internucleotide phosphorothioate dlesters possess sufficient thiol character to form adducts with maleimides. Adenosine triphosphate gamma-S (ATPS) and thymldyl-PS-thymidine (TPST) were each reacted with the reporter molecule N-1 pyrene maleimlde (PM) and the fluoresence intensity was recorded. The observed reactivity of the phosphorothioate nucleotldes towards maleimlde was used as a basis for preparing covalent proteln-nucleottde conjugates of ATPS and of the internucteotkle phosphorothioate diester, deoxyadenylyl-PS-deoxy-adenylyl-PS-deoxyadenosine (dA3(PS)2). The absorb-ance spectra of bovine serum albumin (BSA) conjugates of ATPS and of dA3(PS)2 showed the formation of proteln-nucleotide conjugates, with absorbance maxima near 260 nm. The degree of conjugation was 1.69 nucleotides (nt)/BSA molecule for ATPS and 0.44 nt/BSA molecule for dA3(PS)2. The extent of conjugation of the gamma-S of the nucleoside thlotriphosphate and of the non-bridging sulfur of the internucleotide phosphorothioate diester with malelmide-derivatized protein agreed with their relative reactivity towards PM. Both the gamma-S of the nucleoside thiotriphosphate and the Internucleotide phosphorothioate diester were found to possess sufficient thiol character to permit formation of maleimide-mediated protein conjugates.


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