RELATION BETWEEN THE HIGHER ORDER STRUCTURAL CHANGES OF PROTEIN AND FUNCTION ABNORMARITY OF ABNORMAL HEMOGLOBIN

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It has been recently reported that resonance Raman spectra excited in the UV region at 200-240 can explore the environmental and hydrogen bonding changes of tryptophan (Trp) and tyrosine (Tyr) residues of proteins. we have demonstrated here the quaternary-structural dependent features for Tyr and Trp residues in alpha_1beta_2 interface of hemoglobin (Hb) from 235-nm excited UV resonance Raman (UVRR) spectra. Deoxy Hb A (T-form) showd a UVRR spectrum distinctly different from those of the ligated Hbs (R-form) including osyHb, COHb and NOHb.To characterize the spectral changes of Trp-beta37 at alpha_1beta_2 interface due to the quaternary structure transition, the UVRR spectra of Hb A were compared with the corresponding spectra of Hb Hirose (Trp-beta37->Ser). Comparison of the Hb A - Hb Hirose difference spectra in oxy and deoxy states revealed that the oxygenation- induced changes of Trp RR bands arose mostly from Trp-beta37.The UVRR spectral contribution of alpha42Tyr, Which is located in the "swhitch" region of the alpha_1beta_2 interface and forms an H-bond with the carboxylate side chain of beta99 Asp only in the T-state, was deduced for each of the deoxy- and CO-forms by subtracting the spectra of Hb alpha Y42H from those of Hb A.This suggested that alpha42Tyr is responsible for the frequency shift of Y8a (1617cm^<-1>) and Y9a (1179cm^<-1>) of the Tyr RR bands of Hb alphaY42H upon quaternary structure change are alike.The extent of the oxidation of Hb M Sakatoon and Hb M Boston in the patients blood was determined by measurement of the intensity of EPR signal at g=6.0 for the normal subunits, g=6.7for the mutant subunit of Hb M Saskatoon, and g=6.3 for those of Hb M Boston. About 50% and 76% of mutant subunits in Hb M Boston and Hb M Sakatoon remained reduced in the fresh blood, respectively.