STEADY-STATE FLUORESCENCE ENERGY-TRANSFER MEASUREMENTS OF HUMAN ALPHA-APOHEMOGLOBIN STRUCTURE

A nonfluorescent reagent, 4-phenylazophenylmaleimide 4-PAPM, was att ached to the sole cysteine residue 104(G11) of alpha apohemoglobin ( alpha(0)) and served as an energy acceptor for the single intrinsic tr yptophanyl 14(A12) donor. This novel fluorescence system provided a transmolecular vehicle by which the overall structure of alpha(0) coul d be monitored in 0.05 M potassium phosphate buffer at 5 degrees C. Ra tio of the emission intensities at 335 nm for monomeric solutions (5 x 10(-6)M) of both alpha(0) and alpha(0) 4-PAPM furnished a measure o f the efficiency of energy transfer and average distance of separation (r). An apparent increase in the value of r was observed from pH 6.5 to 8.5, suggesting that the conformation (the structural relationship of the A and G helical segments) of alpha(0) is responsive to its elec trostatic environment. (C) 1994 Academic Press, Inc.