Absorption spectroscopy and circular dichroism studies of sperm whale myoglobin chemically modified on the N-terminal alpha-aminogroup by isothiocyanate reagents.

Abstract

Sperm whale metMb [Mb(SW)] was modified chemically by fluorescein isothiocyanate and methylisothiocyanate. Individual modification products on the α-aminogroup of the N-terminal Val were isolated with ion exchange chromatography (FITC-Mb and MITC-Mb). Absorption spectra in the 200–700 nm region and spectrophotometric titration curves in the Soret band of the modified metMb derivatives and intact metMb were compared. Characteristic differences between them indicate that upon modification there occurs a shift in the equilibrium of isomers of the metMb aquo complex towards the low-spin form. The CD spectra of FITC-metMb and MITC-inetMb in the 200–450 nm region attest to small changes in the heme environment as compared to native metMb without, however, any appreciable conformational changes of the polypeptide chain. No differences have been found in the absorption and CD spectra in the Soret region between native deoxy-Mb and the modified Mb derivatives in deoxy forms. An analysis of the present results and of those reported in the literature shows that the conformational changes at the N-end of Mb upon modification of the N-terminal α-amino group result in structural alterations in the heme environment which are most likely to consist in some reorientation of the side group of His E7 and, possibly, those of phe B14, Phe CD1,and Phe CD4 on the distal side of the heme. A scheme of the electronic conformational interactions (ECI) in ferrimyoglobin is proposed.