CHEMICAL MODIFICATION OF MYOGLOBIN BY ISOTHIOCYANATE REAGENTS - EFFECT OF N-TERMINAL AMINO GROUP MODIFICATION ON PROTEIN CONFORMATION

Sperm whale met-Mb was chemically modified with fluorescein isothiocya nate (FITC) and methyl isothiocyanate (MITC) at pH 6.5-7.0. The isolat ed met-Mb derivatives (FITC-Mb and MITC-Mb) modified at the alpha-NH2- group of Val 1 (NA1) were obtained by ion-exchange chromatography with yields of 10 and 30%, respectively, and characterized. The His 12(A10 ) residue was found to be also thiocarbamylated in MITC-Mb. Large amou nts of remaining intact met-Mb (more than 50%) as well as small fracti ons of more highly modified protein were isolated in both cases. Absor ption and CD spectra of met-Mb, FITC-Mb, and MITC-Mb in the UV- and vi sible regions, spectrophotometric titration of the Soret band, and try ptophan fluorescence of the apo- and holomyoglobins in the pH range 2- 13 were investigated. Modification of the N-terminus was found to have no effect on the conformation (alpha-helicity) of the polypeptide cha in of Mb, but it causes specific changes in the absorption and CD spec tra, pK values of met-hydroxy transition, and pH-dependent fluorescenc e of the modified species compared to native met-Mb. The data suggest both altered local conformation of the N-terminal region and of the he me environment.