Assignment of the 1511 cm −1 UV resonance Raman marker band of hemoglobin to tryptophan
2001; Wiley; Volume: 62; Issue: 3 Linguagem: Inglês
10.1002/bip.1009
ISSN1097-0282
AutoresXiaojie Zhao, Ruopian Chen, Vishwa S. Raj, Thomas G. Spiro,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoAbstract New UV resonance Raman (UVRR) data provide convincing evidence that a characteristic 1511 cm −1 band in the T − R difference spectra of hemoglobin is due to the overtone of the Trp W18 fundamental at 756 cm −1 . Measured isotope shifts for 2‐H and 15‐N substitution at the indole NH group are twice as large for the 1511 cm −1 band as for W18, and the 1511 cm −1 intensity scales with that of W18 in the difference spectrum. Moreover, the UVRR excitation profile of the 1511 cm −1 band tracks that of another tryptophan band, W16. Both are redshifted in hemoglobin, relative to aqueous tryptophan, reflecting H bonding within a hydrophobic environment in the protein. The 2×W18 assignment had been thrown into question by the observation of remnant 1511 cm −1 intensity in the T − R spectra of hemoglobin labeled with tryptophan‐ d 5 , a substitution that shifts W18 over 50 cm −1 . However, reexamination of the data suggests that this remnant intensity may result from a subtraction artifact arising from the downshift of another difference band, W3, from 1549 cm −1 in unlabeled protein to 1522 cm −1 in labeled protein. Restoration of the 2×W18 assignment establishes that the 1511 cm −1 difference band, which is a useful indicator of the extent of T‐state formation in hemoglobin, arises from the same residue, Trpβ37, that gives rise to essentially all of the T − R signal from tryptophan. © 2001 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 62: 158–162, 2001
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