Introduction of a specific binding domain on myoglobin surface by new chemical modification

A new myoglobin, reconstituted with a modified zinc protoporphyrin, having a total of four ammonium groups at the terminal of the two propionate side chains was constructed to introduce a substrate binding site. The protein w ith a positively charged patch on the surface formed a stable complex with negatively charged substrates, such as hexacyanoferrate(III) and anthraquin onesulfonate via an electrostatic interaction, The complexation was monitor ed by fluorescence quenching due to singlet electron transfer from the phot oexcited reconstituted zinc myoglobin to the substrates. The binding proper ties were evaluated by Stern-Volmer plots from the fluorescence quenching o f the zinc myoglobin by a quencher. Particularly, anthraquinone-2,7-disulfo nic acid showed a high affinity with a binding constant of 1.5x10(5) M-1 in 10 mM phosphate buffer, pH 7.0. In contrast, the plots upon the addition o f anthraquinone-2-sulfonic acid at different ionic strengths indicated that the complex was formed not only by an electrostatic interaction but also b y a hydrophobic contact. The findings from the fluorescence studies conclud e that the present system is a useful model for discussion of electron tran sfer via non-covalently linked donor-acceptor pairing on the protein surfac e. (C) 2000 Elsevier Science B.V. All rights reserved.