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Application of α-N-acetylgalactosaminidase and α-galactosidase in AB to O red blood cells conversion.

Artificial cells, nanomedicine, and biotechnology (2012-10-04)
Hongwei Gao, Subo Li, Yingxia Tan, Shouping Ji, Yingli Wang, Guoqiang Bao, Lijuan Xu, Feng Gong
ABSTRACT

Enzymatical conversion of A or B RBCs into group O RBCs (ECORBCs) was achieved by using α-N-acetylgalactosaminidase and α-galactosidase, respectively. Now, we initiated AB to O-RBC conversion by using these two enzymes together. But α-N-acetylgalactosaminidase and α-galactosidase's preserving and their reaction buffer were quite different. The aim of this study is to confirm an available system for converting AB to O RBCs, especially to study the maximal permission amount of PCS which was brought to the system-accompanied enzyme addition. Enzyme activity was detected by using GalNAc-pNp or Gal-pNp as substrates. The efficiency of the conversion of A or B antigen was evaluated by routine method and measured by fluorescence-activated cell sorting analysis. The optimal buffer component and the doses of α-N-acetylgalactosaminidase and α-galactosidase were confirmed according to A and B antigen epitope removal efficiency. The activity of α-N-acetylgalactosaminidase and α-galactosidase was not decreased drastically when they were kept in PCS Buffer in 4°C. The optimal reaction buffer composed of glycine 250 mM and NaCl 3 mM, pH 6.8 and PCS less than 10%(v/v). For converting A(1)B to O RBCs completely, the doses of α-N-acetylgalactosaminidase and α-galactosidase were confirmed as 0.015 mg/ml packed RBCs(pRBCs) for A(1) antigen epitopes and 0.005 mg/ml pRBCs for B epitopes. Approximately 0.004 mg α-N-acetylgalactosaminidase and 0.005 mg α-galactosidase were required to convert 1 ml pRBCs. Our studies indicated that α-N-acetylgalactosaminidase and α-galactosidase were stable in PCS buffer and a modified protocol which was propitious to converting AB to O RBCs was provided.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
α-Galactosidase from green coffee beans, ammonium sulfate suspension, ≥9 units/mg protein
Sigma-Aldrich
α-Galactosidase, positionally specific from Escherichia coli, recombinant, expressed in E. coli, buffered aqueous solution
Sigma-Aldrich
α-Galactosidase I, Alkaline from Cucumis melo

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