Peter Cashion

Purine nucleoside phosphorylase in erythrocytes: Determination of optimum reaction conditions

Reaction conditions for determining the activity of purine nucleoside phosphorylase (PNP; E.C. 2.4.2.1) were investigated. We examined the kinetic parameters for the enzymatic reaction with respect to the substrates inosine and phosphate. We confirmed the pH optimum, established the optimal concentration of xanthine oxidase and that of calcium and magnesium. The Km values for inosine and phosphate were found to be 60 uM and 667 uM, respectively. Optimum assay conditions for PNP activity were established. This optimized method has been compared with other procedures and found to be more sensitive and to yield significantly higher activities. The experimental variation of a manual procedure using these optimum reaction conditions was less than 4.5%. The mean erythrocyte PNP activity of 28 healthy subjects was estimated to be 9.71 U/mL packed cells at 25 degrees C and 18.60 U/mL packed cells at 37 degrees C.

A new colorimetric assay for purine nucleoside phosphorylase

A new colorimetric procedure for the determination of purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1) activity is described. In this procedure, the hydrogen peroxide formed in the PNP-xanthine oxidase reaction is used to oxidize the chromogenic reagents--3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone using the enzyme peroxidase. The rate of enzyme reaction is followed at 520 nm. This procedure correlated well with the UV method (290 nm), with a correlation coefficient of 0.98 (P less than 0.005). Within-run and between-run precision (CV) were less than 2.8% and 3.7%, respectively. Here we also describe an optimized NAD-dependent method (340 nm) for PNP determination. The colorimetric method is superior to both the 340 nm and the UV methods in terms of both sensitivity and precision. The mean erythrocyte PNP activity for 17 healthy subjects was 11.88 U/mL packed cells for the NAD-dependent method and 13.22 U/mL packed cells for the colorimetric method.

Hydrophobic Immobilization of Enzymes and Polynucleotides on Trityl Agarose

It has been reported that agarose can be chemically derivatized to form tritylated [(C6H5)3-C-] agarose (TA) (1–3). The trityl group is attached by a stable ether bond. Depending upon the concentration of trityl groups (10–100 µmol/ml), the resin binds a variety of polynucleotides such as poly A terminated mRNA and denatured DNA as well as most enzymes. Of the 30 or so enzymes tested to date, only pancreatic RNase failed to bind. To maintain specificity polynucleotide binding is best done at lower degrees of tritylation (10–40 µmol/ml) while enzymes are generally immobilized at higher levels (50–100 µmol/ml). From the structures of the respective macromolecules which bind, and from their sensitivity to low dielectric constants, to low ionic strengths, and to chaotropic salts, it is probable that the binding is hydrophobic in nature.