Zhao X, Chen R, Raj V, Spiro T G
Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA.
Biopolymers. 2001;62(3):158-62. doi: 10.1002/bip.1009.
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 2xW18 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 2xW18 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, Trpbeta37, that gives rise to essentially all of the T - R signal from tryptophan.
新的紫外共振拉曼（UVRR）数据提供了令人信服的证据，表明血红蛋白的T - R差光谱中1511 cm⁻¹的特征带是由于色氨酸W18在756 cm⁻¹处基频的倍频。吲哚NH基团上2-H和¹⁵-N取代的测量同位素位移，对于1511 cm⁻¹带来说是W18的两倍，并且1511 cm⁻¹带的强度在差光谱中与W18的强度成比例。此外，1511 cm⁻¹带的UVRR激发谱与另一个色氨酸带W16的激发谱一致。相对于水溶液中的色氨酸，二者在血红蛋白中均发生红移，这反映了蛋白质疏水环境中的氢键作用。用色氨酸-d(5)标记的血红蛋白的T - R光谱中观察到1511 cm⁻¹的残余强度，这使得2×W18的归属受到质疑，这种取代使W18位移超过50 cm⁻¹。然而，对数据的重新审视表明，这种残余强度可能是由于另一个差光谱带W3从未标记蛋白质中的1549 cm⁻¹向下位移到标记蛋白质中的1522 cm⁻¹时产生的减法伪像所致。恢复2×W18的归属表明，1511 cm⁻¹的差光谱带是血红蛋白中T态形成程度的一个有用指标，它来自同一个残基Trpβ37，该残基产生了色氨酸的几乎所有T - R信号。
