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.
O'Malley
S M
SM
Department of Chemistry, University of Massachusetts at Lowell 01854.
McDonald
M J
MJ
D000328:Adult; D000595:Amino Acid Sequence; D001391:Azo Compounds; D003545:Cysteine; D004735:Energy Transfer; D006441:Hemoglobin A / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D046911:Macromolecular Substances; D008301:Maleimides; D013050:Spectrometry, Fluorescence / Q000379:methods; D013053:Spectrophotometry / Q000379:methods; D014364:Tryptophan / Q000032:analysis
D001391:Azo Compounds; D007202:Indicators and Reagents; D046911:Macromolecular Substances; D008301:Maleimides; C086886:4-phenylazophenylmaleimide; D014364:Tryptophan; D006441:Hemoglobin A; D003545:Cysteine