Preparation and the Hæmoglobin Content of Red Cell ‘Ghosts’

Abstract

STUDIES of species differences in red cell phosphatides¹ have led to a re-examination of the problem of standardizing the method of preparing erythrocyte membranes (‘ghosts’, post-hæmolytic residues). It seems essential that if results obtained with physical methods, for example, electron microscopy, are to be correlated with those obtained by chemical analysis, greater uniformity of procedure for preparation of membranes must be sought. The matter has received little recent attention. In what was perhaps the first chemical analysis of the structural protein, stromatin, Jorpes² found his preparations to contain irregular amounts of hæmoglobin, representing 10–30 per cent of the dry weight. Not long afterwards, Adams³ offered spectroscopic evidence for the idea that in the intact cell hæmoglobin is combined with stromatin. Keilin and Hartree⁴, however, were able to show that the finding was explained by the peculiar optical properties of the medium and could not be regarded as an indication of a hæmoglobin–stromatin complex. Since that time the blood-pigment found attached to ‘ghosts’ has come to be regarded, or, at any rate, treated, as a sort of contaminant the physical association of which with the membrane proper interferes with obtaining satisfactory material for analysis of other things. Various methods have been devised for preparing ‘ghosts’, one of which⁵ claims to reduce the iron content to the vanishing point (<0.01 per cent hæmoglobin). It is difficult to find information about the stage in the processing at which the intact membranes begin to break up, or, indeed, what the microscopic appearance of the final product is. Usually, the recommended techniques introduce acid or salt washing at some point to expedite the removal of hæmoglobin—measures which in our hands have led to prompt disintegration of ‘ghosts’, as shown by microscopic examination.