Kinetics of hemoglobin allostery from time-resolved UV resonance Raman spectroscopy: effect of a chemical cross-link

Three kinetic phases along the allosteric reaction path of hemoglobin were determined using ultraviolet resonance Raman (UVRR) difference signals associated with tyrosine acid tryptophan residues. The CO adduct was photolyzed with saturating 419 nm pump pulses, and UVRR spectra were generated at a series of time delays with 229 nm probe pulses. Pump and probe pulses were generated by a pair of 1 kHz, 20 ns Nd:YLF-pumped Ti:sapphire lasers, whose outputs were frequency doubled (pump) and quadrupled (probe), Difference spectra obtained with this system are of better quality than those reported previously, and lead to a better defined time course of the spectral changes associated with the protein motions. Fitting of the time course to successive exponential produces time constants of 0.03, 0.7 and 16 mu s for the intermediates R-deoxy, S and T', in good agreement with previous estimates. A chemically modified hemoglobin, alpha alpha Hb, was also examined. alpha alpha Hb, which is prepared by cross-linking the alpha-chains with bis(3,5-dibromosalicyl) fumarate, is under study as a blood substitute. The time course for alpha alpha Hb was similar to that of unmodified Hb, and the time constants were 0.04, 0.35 and 20 mu s. The first and last of the kinetic phases are essentially unaltered, but the second phase is accelerated by a factor of two. Thus the cross-link speeds up the R-deoxy to S transition, which is proposed to involve re-formation of interhelical H-bonds that are broken in the R-deoxy intermediates via repositioning of the N- and C-terminal helices A and H. This transition may be guided by the cross-link which connects the two alpha-chain G helices in the T conformation. Copyright (C) 2000 John Wiley & Sons, Ltd.