Near-UV Circular Dichroism and UV Resonance Raman Spectra of Individual Tryptophan Residues in Human Hemoglobin and Their Changes upon the Quaternary Structure Transition

• Published in: Biochemistry (Easton) 2012-07, Vol.51 (30), p.5932-5941
• Main Authors: Nagai, Masako, Nagatomo, Shigenori, Nagai, Yukifumi, Ohkubo, Kenichi, Imai, Kiyohiro, Kitagawa, Teizo
• Format: Article
• Language: English
• Subjects: Circular Dichroism - methods; Crystallography, X-Ray - methods; Hemoglobin A - chemistry; Hemoglobin A - genetics; Humans; Hydrophobic and Hydrophilic Interactions; Mutation; Protein Structure, Quaternary; Spectrum Analysis, Raman - methods; Tryptophan - chemistry; Tryptophan - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi300347x

The aromatic residues such as tryptophan (Trp) and tyrosine (Tyr) in human adult hemoglobin (Hb A) are known to contribute to near-UV circular dichroism (CD) and UV resonance Raman (RR) spectral changes upon the R → T quaternary structure transition. In Hb A, there are three Trp residues per αβ dimer: at α14, β15, and β37. To evaluate their individual contributions to the R → T spectral changes, we produced three mutant hemoglobins in E. coli; rHb (α14Trp→Leu), rHb (β15Trp→Leu), and rHb (β37Trp→His). Near-UV CD and UVRR spectra of these mutant Hbs were compared with those of Hb A under solvent conditions where mutant rHbs exhibited significant cooperativity in oxygen binding. Near-UV CD and UVRR spectra for individual Trp residues were extracted by the difference calculations between Hb A and the mutants. α14 and β15Trp exhibited negative CD bands in both oxy- and deoxy-Hb A, whereas β37Trp showed positive CD bands in oxy-Hb A but decreased intensity in deoxy-form. These differences in CD spectra among the three Trp residues in Hb A were ascribed to surrounding hydrophobicity by examining the spectral changes of a model compound of Trp, N-acetyl-l-Trp ethyl ester, in various solvents. Intensity enhancement of Trp UVRR bands upon the R → T transition was ascribed mostly to the hydrogen-bond formation of β37Trp in deoxy-Hb A because similar UVRR spectral changes were detected with N-acetyl-l-Trp ethyl ester upon addition of a hydrogen-bond acceptor.