TY - JOUR
T1 - Heterobifunctional Cross-Linking Agents Incorporating Perfluorinated Aryl Azides
AU - Crocker, Peter J.
AU - Imai, Nobuyuki
AU - Rajagopalan, Krishnan
AU - Boggess, Michael A.
AU - Kwiatkowski, Stefan
AU - Dwyer, Lori D.
AU - Vanaman, Thomas C.
AU - Watt, David S.
PY - 1990/11/1
Y1 - 1990/11/1
N2 - 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%.
AB - 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%.
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U2 - 10.1021/bc00006a008
DO - 10.1021/bc00006a008
M3 - Article
C2 - 2151563
AN - SCOPUS:0025520711
SN - 1043-1802
VL - 1
SP - 419
EP - 424
JO - Bioconjugate Chemistry
JF - Bioconjugate Chemistry
IS - 6
ER -