Abstract
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