Maleimide-mediated protein conjugates of a nucleoside triphosphate gamma-S and an internucleotide phosphorothioate diester.
The purpose of this study was to determine whether the gamma-S of nucleoside thiotriphosphates and the non-bridging sulfur of internucleotide phosphorothioate diesters possess sufficient thiol character to form adducts with maleimides. Adenosine triphosphate gamma-S (ATPS) and thymidyl-PS-thymidine (TPST) were each reacted with the reporter molecule N-1 pyrene maleimide (PM) and the fluorescence intensity was recorded. The observed reactivity of the phosphorothioate nucleotides towards maleimide was used as a basis for preparing covalent protein-nucleotide conjugates of ATPS and of the internucleotide phosphorothioate diester, deoxyadenylyl-PS-deoxy-adenylyl-PS-deoxyadenosine (dA3(PS)2). The absorbance spectra of bovine serum albumin (BSA) conjugates of ATPS and of dA3(PS)2 showed the formation of protein-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 thiotriphosphate and of the non-bridging sulfur of the internucleotide phosphorothioate diester with maleimide-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.
Karim
A S
AS
Division of Biology and Medicine, Brown University, Providence, RI 02912, USA.
Johansson
C S
CS
Weltman
J K
JK
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D015226:Dinucleoside Phosphates / Q000737:chemistry*; D008301:Maleimides / Q000737:chemistry*; D009711:Nucleotides / Q000737:chemistry*; D013455:Sulfur / Q000737:chemistry
D015226:Dinucleoside Phosphates; D008301:Maleimides; D009711:Nucleotides; C043592:maleimide; C022571:adenosine 5'-O-(3-thiotriphosphate); C068420:thymidylyl(3'-5')-5'-thio-5'-deoxythymidine; D013455:Sulfur; D000255:Adenosine Triphosphate