Steady state fluorescence energy transfer measurements of human alpha apohemoglobin structure.

A nonfluorescent reagent, 4-phenylazophenylmaleimide [4-PAPM], was attached to the sole cysteine residue [104(G11)] of alpha apohemoglobin (alpha degree) and served as an energy acceptor for the single intrinsic tryptophanyl [14(A12)] donor. This novel fluorescence system provided a transmolecular vehicle by which the overall structure of alpha degree could be monitored in 0.05 M potassium phosphate buffer at 5(0) C. Ratio of the emission intensities at 335 nm for monomeric solutions (5 x 10(-6) M) of both alpha degree and alpha degree [4-PAPM] furnished a measure of 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 degree is responsive to its electrostatic environment.