Hemes and hemoproteins. 1: Preparation and analysis of the heme-containing octapeptide (microperoxidase-8) and identification of the monomeric form in aqueous solution

Abstract

The heme-octapeptide from cytochrome c, Microperoxidase-8 (MP-8), was prepared by peptic and tryptic digestion of horse heart cytochrome c and purified by gel permeation chromatography in about 50% yield. Conditions for the identification of MP-8 by TLC and analysis by HPLC are described. Study of the concentration-dependence of the absorption spectrum showed that at concentrations of less than or equal to 2.5 X 10(-5) M in aqueous solution at pH 7, 25 degrees C and mu = 0.1, MP-8 exists as an equilibrium mixture of monomers and dimers with KD = 1.17 +/- 0.02 X 10(5) M-1, decreasing to 1.21 +/- 0.02 X 10(4) M-1 and 2.16 +/- 0.21 X 10(3) M-1 in 20% and 50% (v/v) methanol:water mixtures, respectively. Comparison of the Soret region spectrum of monomeric MP-8 with other hemoproteins suggests that it is six-coordinate in aqueous solution with water and His as axial ligands.