Purine nucleoside phosphorylase: purification using an ether-linked formycin B/sepharose 6B resin with unusual properties

Preparative Biochemistry
W W Hall, T A Krenitsky

Abstract

Formycin B [9-deazainosine] was reacted with epoxy-activated Sepharose 6B to form an affinity resin for purine nucleoside phosphorylase (PNPase). This resin had a large capacity (7,600 units/ml) for the enzyme from Escherichia coli. Enzyme retention was dependent on high ionic strength. Although this property is reminiscent of hydrophobic interaction chromatography, analogous resins prepared with pseudouridine or monoethanolamine instead of with formycin B, did not retain the enzyme even at high ionic strength. Furthermore, hypoxanthine facilitatted elution of the enzyme from the resin. It appeared, therefore, that the enzyme was not bound simply by hydrophobic interactions. A simple two-step purification procedure for PNPase from Escherichia coli was devised using this resin. Overall recovery was 50%, and purity of the final preparation was greater than 95%. This resin was also useful in the purification of PNPase from human erythrocytes. The ether linkage between formycin B and Sepharose 6B, together with the carbon-to-carbon linkage between the pentose and heterocyclic moieties of formycin B, provided stability to both chemical and enzymatic degradation. After 5 years of use and exposure to a variety of biological preparatio...Continue Reading

References

May 15, 1986·Archives of Biochemistry and Biophysics·T A KrenitskyW W Hall
Mar 1, 1997·European Journal of Cancer Care·G H Knowles, D I Jodrell
Apr 9, 1954·Science·H M KALCKAR

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