Optimized procedures for investigating changes in human erythrocyte membrane proteins using treatment with calcium...

In order to establish reproducible sensitive techniques for investigating diseases in which membrane defects have been implicated, human red cell membrane proteins were studied by SDS electrophoresis in ghosts prepared from the blood of 35 separate adult donors using treatment with 1 mmol/l calcium and proteolytic dissection under standardized conditions. The effects of calcium were consistent with increased binding of cytosolic proteins in membranes from fresh blood and endogenous proteolysis in membranes from aged blood. Exogenous proteolysis was most effective with trypsin and was facilitated both by pre-binding this protease to the membrane and by inclusion of 1 mmol/l Ca2+. The nature and extent of these effects was highly dependent on the storage time of the blood. Preliminary indications of cleavage fragment length polymorphism was obtained after treatment with the S. aureus V8 protease but not after trypsin or alpha chymotrypsin treatment.