A number of methods for the assay of n-ribosed-phosphate ketolisomerase (ribosephosphate isomerase EC 5.3.1.6) have been reviewed by Knowles et al. (1). A procedure employed in this laboratory has been described (2) in which, using n-ribose 5-phosphate as a substrate, the oxidation of NADH was followed continuously at 340 rnp after the addition of n-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1)) transketolase (EC 2.2.1.1) and a mixture of glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) and triosephosphate isomerase (EC 5.3.1.1). Details of a related assay for n-ribulose-5-phosphate 3-epimerase were also published (2).
The above assays have a number of disadvantages. The ribosephosphate isomerase assay at 340 rnp involves three consecutive auxiliary reactions to couple the isomerase reaction to NADH oxidation and a considerable excess of two enzymes that are not commonly available. The assay for the epimerase requires n-ribulose '5-phosphate, a substrate that is expensive and difficult to isolate. The commonly used calorimetric procedure of Dische and Borenfreund (3) suffers from drawbacks due to variations in the depth of color given by a standard quantity of ribulose 5-phosphate and interference by the presence of n-ribulose-5phosphate 3-epimerase. These disadvantages are overcome in the method of Bruns et al. ( 4) in which the disappearance of n-ribose 5-phosphate is followed using the phloroglucinol reaction ( 5), but, even with careful technique, considerable variations in the depth of color given by a standard amount of ribose 5-phosphate were found.
The method for n-ribose-5-phosphate ketol-isomerase described here is based upon the observation of Knowles and Pon ( 6) that an absorption band with a peak at 280 rnp appears when the enzyme is mixed with n-ribose 5-phosphate. It is similar to the assay at 286 mp described recently by Knowles et al. (1) except that a wavelength of 290 rnp is
Cobrimetric
Measurements.