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Kinetics of hemoglobin allostery from time‐resolved UV resonance Raman spectroscopy: effect of a chemical cross‐link
Author(s) -
Zhao Xiaojie,
Balakrishnan Gurusamy,
Moore Edwin G.,
Spiro Thomas G.
Publication year - 2000
Publication title -
journal of raman spectroscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.748
H-Index - 110
eISSN - 1097-4555
pISSN - 0377-0486
DOI - 10.1002/(sici)1097-4555(200004)31:4<349::aid-jrs548>3.0.co;2-6
Subject(s) - chemistry , raman spectroscopy , kinetics , resonance (particle physics) , hemoglobin , resonance raman spectroscopy , reaction rate constant , kinetic energy , adduct , photodissociation , analytical chemistry (journal) , photochemistry , organic chemistry , atomic physics , optics , physics , quantum mechanics
Three kinetic phases along the allosteric reaction path of hemoglobin were determined using ultraviolet resonance Raman (UVRR) difference signals associated with tyrosine and 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 µs for the intermediates R deoxy , S and T′, in good agreement with previous estimates. A chemically modified hemoglobin, α αHb, was also examined. α αHb, which is prepared by cross‐linking the α‐chains with bis(3,5‐dibromosalicyl) fumarate, is under study as a blood substitute. The time course for α αHb was similar to that of unmodified Hb, and the time constants were 0.04, 0.35 and 20 µs. 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 α‐chain G helices in the T conformation. Copyright © 2000 John Wiley & Sons, Ltd.