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 tryptoph anyl 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, p H 7.0, at 5 degrees C, and the structural flexibility of alpha-apohemo globin, in the absence and presence of human beta-heme-containing chai ns, was examined by a steady-state solute quenching technique. The que nched efficiencies (E(Q)) and Forster distances (R(0)(Q)) were analyze d by least-squares to determine the goodness of fit (chi(R)(2)) for th e assumed distribution parameters: average distance (r) over bar and h alf-width hw. Data for alpha-apohemoglobin in the absence and presence of beta(h) chains yielded values for (r) over bar of 18 and 22 Angstr om and hw of 20 and 8.5 Angstrom, respectively. Although the increase in (r) over bar for alpha-apohemoglobin in the presence of beta(h) cha ins 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 demon strated here may be an important aspect of human hemoglobin assembly.