Assignment of the 1511 cm(-1) UV resonance Raman marker band of hemoglobinto tryptophan

New UV resonance Raman (UVRR) data provide convincing evidence that a chara cteristic 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 iso tope shifts for 2-H and 15-N substitution at the indole NH group are twice as large for the 1511 cm-l band as for W18, and the 1511 cm(-1) intensity s cales with that of W18 in the difference spectrum. Moreover, the UVRR excit ation profile of the 1511 cm(-1) band tracks that of another tryptophan ban d, W16. Both are redshifted in hemoglobin, relative to aqueous tryptophan, reflecting H bonding within a hydrophobic environment in the protein. The 2 xW18 assignment had been thrown into question by the observation of remnan t 1511 cml intensity in the T - R spectra of hemoglobin labeled with trypto phan-d(5), a substitution that shifts W18 over 50 cm(-1). However, reexamin ation of the data suggests that this remnant intensity may result from a su btraction artifact arising from the downshift of another difference band, W 3, from 1549 cm(-1) in unlabeled protein to 1522 cm(-1) in labeled protein. Restoration of the 2xW18 assignment establishes that the 1511 cm(-1) diffe rence band, which is a useful indicator of the extent of T-state formation in hemoglobin, arises from the same residue, Trp beta 37, that gives rise t o essentially all of the T - R signal from tryptophan. (C) 2001 John Wiley & Sons, Inc.