Assignment of the 1511 cm(-1) UV resonance Raman marker band of hemoglobin to tryptophan.
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.
Zhao
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Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA.
Chen
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R
Raj
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Spiro
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TG
D000328:Adult; D003903:Deuterium; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D066298:In Vitro Techniques; D009584:Nitrogen / Q000737:chemistry; D013059:Spectrum Analysis, Raman; D014364:Tryptophan / Q000737:chemistry
D006454:Hemoglobins; D006859:Hydrogen; D014364:Tryptophan; D003903:Deuterium; D009584:Nitrogen