Spin-label and fluorescence labeling studies of the thioester bonds in human alpha 2-macroglobulin.
Upon cleavage of the reactive thioester bonds (Cys-949-Glx-952) of tetrameric human alpha 2-macroglobulin (alpha 2M) by methylamine, one sulfhydryl group per alpha 2M subunit is exposed. These identical sulfhydryl group sites were labeled with the thiol-specific nitroxide spin-labels (1-oxy-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methyl methanethiosulfonate and (1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)methyl methanethiosulfonate, a homologous series of maleimide spin-labels, and the thiol-specific fluorescent probe 2-[(4-maleimidophenyl)amino]naphthalene-6-sulfonic acid sodium salt (MANS). The ESR and fluorescence results showed that these sulfhydryl group sites were at the base of a narrow crevice that is greater than or equal to 8 A deep. Although the bound MANS fluorophore was slightly blue shifted with an enhanced quantum yield vs the free label in water, the environment of the sulfhydryl site appeared to be of a polar nature when compared with the emission maxima in several solvents of varying polarity. The Glx residue participating in the thioester linkage in the intact protein was labeled with 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl. The distance between the Glx and Cys moieties was estimated at greater than or equal to 10-25 A from double spin-labeling experiments.
Zhao
B L
BL
Department of Chemistry, Ohio State University, Columbus 43210.
Musci
G
G
Sugawara
Y
Y
Berliner
L J
LJ
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D008744:Methylamines / Q000378:metabolism; D013113:Spin Labels / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D008744:Methylamines; D013113:Spin Labels; D000511:alpha-Macroglobulins; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; D004228:Dithionitrobenzoic Acid; C027451:methylamine