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 asso ciated with tyrosine acid tryptophan residues. The CO adduct was photolyzed with saturating 419 nm pump pulses, and UVRR spectra were generated at a s eries of time delays with 229 nm probe pulses. Pump and probe pulses were g enerated by a pair of 1 kHz, 20 ns Nd:YLF-pumped Ti:sapphire lasers, whose outputs were frequency doubled (pump) and quadrupled (probe), Difference sp ectra obtained with this system are of better quality than those reported p reviously, and lead to a better defined time course of the spectral changes associated with the protein motions. Fitting of the time course to success ive exponential produces time constants of 0.03, 0.7 and 16 mu s for the in termediates R-deoxy, S and T', in good agreement with previous estimates. A chemically modified hemoglobin, alpha alpha Hb, was also examined. alpha a lpha Hb, which is prepared by cross-linking the alpha-chains with bis(3,5-d ibromosalicyl) fumarate, is under study as a blood substitute. The time cou rse for alpha alpha Hb was similar to that of unmodified Hb, and the time c onstants were 0.04, 0.35 and 20 mu s. The first and last of the kinetic pha ses are essentially unaltered, but the second phase is accelerated by a fac tor of two. Thus the cross-link speeds up the R-deoxy to S transition, whic h is proposed to involve re-formation of interhelical H-bonds that are brok en 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 conn ects the two alpha-chain G helices in the T conformation. Copyright (C) 200 0 John Wiley & Sons, Ltd.