A Hemochromogen Component of Liver Microsomes

Abstract

Submicroscopic particles (microsomes) isolated from the livers of various animals by means of differential centrifugation are obtained in the form of a transparent jelly with a distinct amber color." 2 The nature of the coloring matter of these particles has not been thoroughly investigated. The fact that microsomes possess little oxidative enzyme ac-tivity2 suggests that the amber hue is not due to the normal cytochrome pigments associated with biological oxidations. Bensley3 has extracted with lipid solvents from microsomes a red colored material which he suggests is lipid in nature. In this communication we wish to present evidence that the major intrinsic pigment of liver microsomes is a hemochromogen compound resembling, but distinct from, the well known cytochromes a, b and c first described by Keilin.4 Materials and Methods.-The tissue used in these studies was rat liver, perfused in situ with cold isotonic saline to remove blood pigments which might otherwise contaminate the homogenate prepared from it. The perfused liver was homogenized in 9 volumes of ice-cold 0.25 M sucrose, and the homogenate fractionated by differential centrifugation at 0-50C.5 The microsome fraction so obtained presented a typical transparent, dark amber appearance. Finally, to wash away pigments that might be merely adsorbed, the microsome pellets were rehomogenized in ice-cold isotonic saline and recovered by high-speed centrifugation. For the spectrophotometric studies, washed microsome pellets were treated with a solution of 1% Na desoxycholate in 0.05 M glycylglycine buffer, pH 7.0 in a manner similar to that described by Ball, Strittmatter and Cooper6 for a heart muscle preparation. The slightly turbid preparation obtained was centrifuged at 110,000 times the force of gravity for 15 minutes at 0-5o and the clear orange fluid removed by pipette from between a minute-colorless precipitate and a white scum-like surface layer. These desoxycholate preparations were prepared so that they contained the microsomal material from 0.5 g. of liver in each milliliter of solution. Samples of this microsome preparation were placed in special vessels consisting of a Thunberg tube fused to a standard Beckman corex cuvette. When atmospheres other than air were required, the vessels were evacuated 3 times, flushed and equilibrated with the desired gas. Substrates, if any, were tipped in from the hollow Thunberg stopper, and absorption measurements made in a Beckman model DU spectrophotometer at room temperature. Readings were made every 1 or 2 m,u in regions of maximal 19