A Simple, Rapid Method for the Multiple Assay of Tryptophan Pyrrolase in Drosophila

This report describes the development of a simple, rapid method for the kinetic assay of tryptophan pyrrolase in Drosophila. By means of this method, one can readily make simultaneous measurements of tryptophan pyrrolase activity in as many as 36 samples. INTRODUCTION Tryptophan pyrrolase is essential in Drosophila for the formation of eye pigments. Vermilion eye mutants, for example, have low levels of tryptophan pyrrolase activity (Baglioni, 1960; Kaufman, 1962; Marzluf, 1965). Although an assay for tryptophan pyrrolase in Drosophila has been reported (Kaufman, 1962), it involves a modification of a diazotization procedure (Bratton and Marshall, 1939) whereby kynurenine is measured after the reaction has been stopped with acid. Because this is cumbersome and time-consuming, we decided to develop a method that was both simpler and more rapid. We also investigated the components of the system with the intention of developing an optimal reaction mixture. The result of these studies was the development of a simple, rapid method for the multiple assay of tryptophan pyrrolase in Drosophila. This assay, as well as the one previously reported, is based on the following principle. Tryptophan pyrrolase converts L-tryptophan to N-formyl kynurenine. The latter is subsequently converted to kynurenine by a second enzyme, kynurenine formamidase. Because kynurenine formamidase occurs in Drosophila in great excess (Glassman, 1956), the amount of kynurenine formed can be used as an index of tryptophan pyrrolase activity. METHODS Wild-type Drosophila larvae were homogenized in 3 volumes of a 0.14 M KC10.0025 N KOH medium. The resulting homogenate was centrifuged at 15,000 x g for 15 minutes. The supernatant was decanted and used as the source of the enzyme in the reaction mixture. The reaction mixture contained, in final concentrations: 7 micromoles/ml of L-tryptophan; 0.00125 mg/ml of hematin; 10 micromoles/ml of sodium ascorbate; and 110 micromoles/ml of sodium phosphate buffer, pH 7.0. The reaction mixture also contained 1.0 ml of the 15,000 x g supernatant and water. The final volume was adjusted to 4.53 ml with water so that the values of optical density at 365 m/i would be equal to the concentration of kynurenine in micromoles/ml. The solutions of tryptophan, hematin, and ascorbate were prepared fresh weekly, adjusted to a pH of 7.0, and stored at 5°C. The reaction mixture was incubated at 37°C in open 10 ml DeLong culture flasks on a Dubnoff shaker. The use of a Dubnoff shaker with racks made possible the simultaneous incubation of up to 36 flasks. At intervals of 30 minutes, the mixture in each flask was decanted into a quartz cuvette, and the amount of kynurenine present was determined by absorbancy measurements at 365 ran against water. The mixture was then decanted back into the same flask, and the flask was returned to the incubator. Because the entire process required less Part of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at The Ohio State University. Manuscript received January 11, 1968. Present address: P. O. Box 8096, Cincinnati, Ohio 45208. THE OHIO JOURNAL OF SCIENCE 68(6): 285, November, 1968. 286 WILLIAM W. MORRISON AND WALTER J. FRAJOLA Vol. 68 than one minute, the absorbancy of the contents of 30 flasks could be measured every 30 minutes. By incubating for a period of three hours, it was thus possible to obtain six 30-minute determinations for each of the 30 flasks. The reaction was no longer linear after three hours, so the measurements were discontinued at that time. Because all reaction mixtures were measured in the same cuvette, no window corrections were necessary. The values obtained for "no tryptophan" blanks were subtracted from the experimental values to determine the amount of kynurenine formed. Tryptophan pyrrolase activity was expressed as micromoles of kynurenine formed per hour per milliliter of the 15,000 x g supernatant. RESULTS From figure 1 it is evident that kynurenine has an absorption maximum at 365 m/j,. It is also evident that L-tryptophan does not absorb light at this wavelength, even in concentrations 100 times as great.