Chemical modification of myoglobin by isothiocyanate reagents. the effect of modifying the N-terminal amino group on protein conformation
Postnikova, G.B.
Biokhimiia 59(10): 1445-1457
1994
ISSN/ISBN: 0320-9725 PMID: 7819386 Document Number: 422982
Sperm whale met-Mb was chemically modified by fluorescein isothiocyanate (FITC) and methylisothiocyanate (MITC) at pH 6.5-7.0. Individual met-Mb derivatives, FITC-Mb and MITC-Mb, modified at the alpha-NH2-group of Val I (NAI), were obtained by ion exchange chromatography with 10 and 30% yields, respectively, and characterised. The His 12 (AI0) residue was found to be additionally thiocarbamylated in MITC-Mb. Large amounts of the remaining intact met-Mb (more than 50%) were isolated in both cases alongside with small fractions of the deeper modified protein. The absorption and CD spectra of met-Mb, FITC-Mb and MITC-Mb in the UV and visible spectral regions, the spectrophotometric titration curves in the Soret band and tryptophanyl fluorescence of the apo- and holomyoglobins in the pH range of 2-13 were investigated. It is shown that modification of the N-end had no influence on the conformation (alpha-helicity) of the polypeptide chain of Mb but caused specific changes in the absorption and CD spectra, pK values of met-hydroxy transition, and the pH-dependent fluorescence of the modified species as compared to the native met-Mb. The data obtained evidence in favour of both the changed local conformation of the N-terminal region and the heme environments in FITC-Mb and MITC-Mb.