Preparation of Crystalline Cytochrome c from Beef Heart Muscle.

Abstract

A simple method for the crystallization of beef heart cytochrome c is described. Amino acid analysis together with a number ofphysico-chemical characteristics of the preparation are reported. An explana-tion for previous discrepancies in the sulphur content of cytochrome c is given. F or some years we have been concerned with the development of a simple, reproducible method 1 of preparing crystalline mammalian cytochrome c *. As early as 1950 we introduced a new chromatographic procedure 2 in the preparation of cytochrome c. This involved the use of the ion-exchange resin Amberlite IRC-50 and much of the procedure to be described is based on the use of this carboxylic acid type resin. Japanese biochemists 3 -G who have recently carried out a great deal ofwork with cytochrome c from a variety of sources have taken advantage of this ion exchange treatment during the purification and concentration procedure. The acid extraction of cytochrome c was introduced by Theorell 7 and was also employed in the classical modifica-tions by Keilin and Hartree s, 9 • The latter workers, however, used trichloro-acetic acid both for the extraction and the final precipitation of the pigment. There has been some question, however, as to the effect of TCA on cyt.c and as we 10 had already found H 2S04 to give a better yield in the extraction of salmon heart cyt.c this acid was accordingly used for the extraction of beef heart cyt.c. EXPERIMENTAL Assay method. The concentration of cyt.c was determined spectrophotometrically after reduction with sodium dithionite in 0.065 M phosphate buffer, pH 6.8 using Ei % 550 mµ = 22.7, corresponding to cytochrome c of 0.435 % iron content. * The following abbreviations will be used: cyt.c = cytochrome c, TCA = trichloroacetic acid. (A) Amberlite. 1/2 kg Amberlite XE-64 was suspended in 1J12NNaOH11.nd allowed to stand in a water-bath (50°) for 6 h "'ith occasional stirring. The supernatant was discarded and the resin washed with distilled water until neutral. 51 of 5 % H 2SO, were then added and the mixture after stirring allowed to stand for l /2 h. The resin was next washed with distilled water until neutral and then suspended in 5 1 of 5 % ammonium hydroxide and allowed to stand for 1 h with occasional ·stirring. Finally the resin was washed with redistilled water until neutral and filtered on a Büchner funnel until nearly dry (20 % moisture content). The resin was stored in this state. (B) Duolite. 10 g of Duolite CS-101 was ground by hand using a pestle and mortar and suspended in 200 ml 2.5 % H 2S04 • lt was then washed with distilled water until neutral; at the same time the finest particles were removed by decantation. The ammo-nium salt of the resin was formed by standing for l /2 h with occasional stirring in 250 ml of 5 % ammonium hydroxide. lt was then washed with distilled water until neutral. The regeneration procedure was repeated once, using redistilled water instead of distilled water as formerly. lt was filtered and stored damp es for the Amberlite. Preparation and purification of cytochrome c Step 1. 6 kg fresh cow hearts were minced, 15 l of cold distilled water added and the pH adjusted to 4.1 with 1 N H 2SO,. This and the subsequent steps were carried out at 4°. The mixture was allowed to stand for about 1 h with occasional stirring. After filtra-tion through cheese cloth the mince was extracted further with 6 1 of water. The total extract was brought to pH 7 with 4 N ammonia, allowed to stand for 1 % h, then