| Ruminococcus gnavus is a Gram positive, non spore-forming obligate anaerobe; found as a normal part of the human digestive flora. In culture, this organism has been shown to produce an α 1–3 galactosidase. We have demonstrated this enzyme can be fractionated, characterized, and shown to specifically hydrolyze the type B epitope on erythrocyte membranes. This results in the seroconversion of human erythrocyte membranes from type B to type O.; An α 1–3 galactosidase from R. gnavus was highly purified using a variety of low-pressure column chromatographic techniques, as well as HPLC. An ELISA based assay was developed and used throughout the purification process to specifically screen for α 1–3 galactosidase activity against type B membrane epitopes.; Characterization of highly purified enzyme preparations included determination of pH optimum and enzyme stability, ionic strength optimum, temperature optimum, substrate specificity, and buffer effects. Molecular weight was determined by gel filtration chromatography, and SDS-PAGE. The effect on enzyme activity in several different red blood cell preservative solutions and type B plasma, was also investigated. Conversion of the type B epitope to type H (blood type O) after exoglycosidase treatment was examined. Optimal reaction conditions for overall seroconversion of type B cells to type O were explored.; Results of these studies have established the capability of the R. gnavus α-galactosidase to remove the terminal galactose from the type B epitope, resulting in a conversion to H epitope (type O antigen). A purified exoglycosidase specific for the seroconversion of type B to O erythrocytes would have numerous useful applications. Of immediate importance is the possibility of converting antigenic epitopes on entire unit volumes of red blood cells. This type of technology would result in the ability to convert erythrocyte type B in order to meet immediate demand for universally transfusable type O red blood cell units. This would result in an overall increase in the numbers of type O units available for transfusion, and would allow for greater flexibility and efficiency in the management of blood bank inventories. |
