[Chemical modification of myoglobin by isothiocyanate reagents. The effect of modifying the N-terminal amino group on protein conformation].
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.
Postnikova
G B
GB
D000818:Animals; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D007202:Indicators and Reagents; D017879:Isothiocyanates / Q000737:chemistry*; D008786:Metmyoglobin / Q000737:chemistry*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014907:Whales
D007202:Indicators and Reagents; D017879:Isothiocyanates; D008786:Metmyoglobin; D016650:Fluorescein-5-isothiocyanate; C005227:methyl isothiocyanate
Khimicheskaia modifikatasiia mioglobina izotiotsianatnymi reagentami. Vliianie modifakatsii N-kontsevoĭ aminogruppy na konformatsiiu belka.