Del Vecchio, V. G.

Fuscaldo, K. E.

Kaczmarczyk, W. K.

Fungal Genetics Reports

English

Kansas State University

Fungal Genetics Reports Volume 3 Article 8 A rapid assay for tyrosinase activity K. E. Fuscaldo W. K. Kaczmarczyk V. G. Del Vecchio Follow this and additional works at: https://newprairiepress.org/fgr This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Recommended Citation Fuscaldo, K. E., W.K. Kaczmarczyk, and V.G. Del Vecchio (1963) "A rapid assay for tyrosinase activity," Fungal Genetics Reports: Vol. 3, Article 8. https://doi.org/10.4148/1941-4765.2154 This Technical Note is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Fungal Genetics Reports by an authorized administrator of New Prairie Press. For more information, please contact cads@k-state.edu. A rapid assay for tyrosinase activity Abstract A rapid assay for tyrosinase activity This technical note is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol3/iss1/8 Fuscaldo, K.E., W. K. Kaczmorczyk, The enzyme, tyrosinose, has been extensively used and V. G. Del Vecchio. A rapid assay for in studies of the genetic control of enzyme synthesis. tyrosinase activity. It is widely distributed in plant, animal, fungal and bacterial cells. Tyrosinose catalyzes the oxidation of tyrosine or dihydroxyphenylalanine (dopa) in the biosynthesis of melanin. The enzyme, in the presence of substrate results in the formation of an insoluble block pigment which con be quantitatively assayed spectrophotometrically. A rapid, simplified method which con be semi-quantitated has been used for the detection of this enzyme in extracts of Neurospora crosso and Bacillus subtilis. This method, o modif:cation of one develop- ed by Cooper and Brow-es not require extensiveinstrumentation and is useful for rapid screening procedures, since extracts of several strains con be tested and cornpored simultaneously. Essentially, the method consists of adding extracts of disrupted cells to wells cut in on agar plate into which substrate has been incorporated. The presence of the enzyme is evidenced by o ring of block pig- ment which precipitates in the ogar surrounding the well. Preliminary results indicate that this assoy may be valuable for semi-quantitative determinations. The size of the block ring and the density of the pigment granules surrounding the well is proportional to the concentration of the extract used in the test and to the degree of purification of the enzyme. The prolonged lag period usually encountered when tyrosine is used OS the substrate con be minimized by using partially purified extracts and by incorporating o low concen- tration of dopa along with the tyrosine in the agar. The enzyme has on absolute requirement for copper. The agar used in the petri dishes must be carefully prepared to ovoid the auto-oxidation of the dopa. Two solutions are mode up separately and combined iust prior to pouring the plates. 8.0 g. of lonager ore added to 500.0 ml of phosphate buffered soline (0.85% NaCl buffered at pH 6.8 with 0.5 M phosphate) and autoclaved. A second solution containing o I:3 ratio of dopo to tyrosine is prepared by adding 0. I g. dopa and 0.3 g. tyrosine to 500 ml of distilled water which was made acidic with 4 drags of 6 N HCI. The dopa-tyrosine solution was added to the buffer when the latter hod cooled to 65-70 C. 30 ml aliquots were dispensed to petri dishes ond allowed to solidify. Wells were cut in the ogor using standard tem- plates available for the preparation of agar-diffusion plates. The bottom of each well was sealed with o drop of agor prior to use. The enzyme extracts from Bacillus subtilis require both the presence of copper and dopa inagor for good results. The catalytic octivityfmurospora enzyme con be demonstrated without the addition of exogenous copper. (Supported in part by o U.S. P. H. 5. Grant, N. 1. H. A-5376,2 Dr. K. E. Fuscaldo) ---Deportment of Biology, St. John’s University, Jamaica, New York, U.S.A. Klingmijller, W. Growing Neurospora A method has been developed for growing individual colonies attached too gloss surface in colonies of Neurospora adhering to gloss surfacer liquid medium. flooded with liquid medium. Fries minimal medium is prepared without sutro~e or ogar. At the home time 0. I% SOIUtiOnS of sorbose and fructose ore prepared seporotely and filter-sterilized. Up to 500 conidio ore added to 100 ml of the final mixed medium and the suspension is distributed into 5 petri dishes. Growth in this medium is colonial, the colonies sticking to the bottom of the dishes, submerged in the medium. Growth is slow, dependin on the strain used. J With the wild type (74 A) colonies con be checked and counted after 7 days at 25 C at which time they ore still very vnoll. Mutants hove been pro- duced by nitrous acid treatment that grow foster; others, that do not grow ot o/I, Growth is nearly independent of the quotient fructose/sorbose in the range from 0.01% to 0. 25% fructose combined with 0. I% sorbose. This is exemplified in the following table for two strains (oper~otionol numbers S+3/1 and S+3/3): 

A rapid assay for tyrosinase activity

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