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

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 ≤2.5 × 10-5 M in aqueous solution at pH 7, 25°C and μ = 0.1, MP-8 exists as an equilibrium mixture of monomers and dimers with KD = 1.17 ± 0.02 × 105 M-1, decreasing to 1.21 ± 0.02 × 104 M-1 and 2.16 ± 0.21 × 103 M 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. © 1986.