Heterobifunctional cross-linking agents incorporating perfluorinated aryl azides.
New heterobifunctional cross-linking reagents were developed that possess a photoactive tetrafluorinated phenyl azide as the photoactive terminus and a chemically reactive succinimidyl ester as the electrophilic terminus. These reagents, succinimidyl N-(4-azido-2,3,5,6-tetrafluorobenzoyl)tyrosinate (9) and succinimidyl 2-(4-azido-2,3,5,6-tetrafluorophenyl)thiazole-4-carboxylate (15), were designed to possess either an 125I or 35S radiolabel, respectively. In a biochemical study, the latter reagent was coupled to Lys-75 of calmodulin (CaM), and the radioiodinated monoadduct was photochemically cross-linked, in a calcium-dependent manner, to the porcine erythrocyte plasma membrane Ca2+, Mg2(+)-ATPase. Densitometry scans of the gel indicated a reproducible 22% cross-linking of the CaM with one of the Ca2+,Mg2(+)-ATPase bands. Since the purification of the Ca2+,Mg2(+)-ATPase results in micelles having Ca2+,Mg2(+)-ATPase with its CaM binding site oriented both to the inside and outside of the micelle, the amount of Ca2+,Mg2(+)-ATPase available for cross-linking was reduced by approximately half, suggesting that the actual cross-linking efficiency was on the order of 40%.
Crocker
P J
PJ
Department of Chemistry, University of Kentucky, Lexington 40506.
Imai
N
N
Rajagopalan
K
K
Boggess
M A
MA
Kwiatkowski
S
S
Dwyer
L D
LD
Vanaman
T C
TC
Watt
D S
DS
D000818:Animals; D001386:Azides / Q000138:chemical synthesis* / Q000737:chemistry; D017301:Ca(2+) Mg(2+)-ATPase / Q000097:blood / Q000737:chemistry; D002118:Calcium / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000097:blood / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000138:chemical synthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000201:enzymology; D015394:Molecular Structure; D010777:Photochemistry; D013056:Spectrophotometry, Ultraviolet; D013552:Swine; D013844:Thiazoles / Q000138:chemical synthesis*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry
D001386:Azides; D002147:Calmodulin; D003432:Cross-Linking Reagents; D013844:Thiazoles; C069580:succinimidyl N-(4-azido-2,3,5,6-tetrafluorobenzoyl)tyrosinate; C069581:succinimidyl 2-(4-azido-2,3,5,6-tetrafluorophenyl)thiazole-4-carboxylate; D014443:Tyrosine; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium