Monitoring the effect of subunit assembly on the structural flexibility of human alpha apohemoglobin by steady-state fluorescence.
A single energy transfer distance, between the sole intrinsic tryptophanyl donor [14(A12)] and a nonfluorescent sulfhydryl acceptor probe (4-phenylazophenylmaleimide, PAPM) attached to the only cysteine [104(G11)], has been employed to examine the effect of subunit assembly on the structure of the heme-free human alpha-hemoglobin. Efficiencies of energy transfer were measured in 0.05 M potassium phosphate buffer, pH 7.0, at 5 degrees C, and the structural flexibility of alpha-apohemoglobin, in the absence and presence of human beta-heme-containing chains, was examined by a steady-state solute quenching technique. The quenched efficiencies (EQ) and Förster distances (R0Q) were analyzed by least-squares to determine the goodness of fit (chi R2) for the assumed distribution parameters: average distance r and half-width hw. Data for alpha-apohemoglobin in the absence and presence of beta h chains yielded values for r of 18 and 22 A and hw of 20 and 8.5 A, respectively. Although the increase in r for alpha-apohemoglobin in the presence of beta h chains was presumably a consequence of additional quenching from the heme moiety, the change in the half-width strongly indicated a decrease in the flexibility of the alpha-apohemoglobin chain within the assembled protein. A transition in structural flexibility similar to that demonstrated here may be an important aspect of human hemoglobin assembly.
Department of Chemistry, College of Arts and Sciences, University of Massachusetts at Lowell 01854.
S M
SM
O'Malley
M J
MJ
McDonald
D000328:Adult; D005453:Fluorescence; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D011487:Protein Conformation
D006454:Hemoglobins