A New Substrate for Fluorometric Determination of Oxidative Enzymes SIR: In 1963 Sharman (I) reported the use of ferricyanide as an oxidant to determine homovanillic acid fluorometrically, and recently Sat0 (2) and Corrodi (3) reversed this and used homovanillic acid fix the quantitative determination of ferricyanide. The compound(1) is converted upon oxidation to the highly fluorescent c:ompound(II), which has a A, of 315 mp and a ,A, of 425 mp. Preliminary results indicated that homovanillic acid can be used as a substrate for the sensitive determination of oxidaf ive enzymes, such as peroxidase. The homovanillic acid solutions appear very stable, and thus would be a distinct adiantage over the diacetylfluorescin method of Keston and Brandt (4), or the procedure of Andreae (5)in which the disappearance of the fluorescence of scopoletin upon oxidation is observed.
Table I. Variation of Rate of Oxidation of Homovanillic Acid (AFjmin) as a Function of Peroxidase and Peroxide Concentration [Peroxidase], units/ml [Peroxide], M AF/min 0 3 x 10-3 0.005 3 x 10-3 3 x 10-3 3 x 10-3 3 x 10-3 3 x 10-8 1.5 X 10-6 3 x 10-7 1 x 10-7 0
0.00600 0.0229 0.110 0.229 2.3 2.3 2.3 2.3 0
0.19 0.72
3.46 7.30 3.2 1.6 0.33 0.11
O.Oo0
YHzCOOH
+ H202 + Peroxidase
Me0
--+
Me0
@ @
OMe
I
I
OH
OH 1
Non-Fluorescent
OH I1 Fluorescent
+ 2 HzO
EXPERIMENTAL
Solutions. Homovanil iic acid solutions were prepared by dissolving the CP compound (Calbiochem. Co.) in distilled water. PEROXIDASE. A stock 0.5 mg per ml solution of horse radish peroxidase (Calbicichem. Co., Los Angeles) was prepared by dissolving 5 m,: of enzyme in 100 ml of distilled water. The enzyme was assayed (6) and was found to contain 137 units per mg. HYDROGEN PEROXIDE.A 0.3% solution was prepared by diluting a 30% stock solution of hydrogen peroxide (Merck and Co., Rahway, N. J.) with distilled water. Apparatus. All fluorescence measurements were made with an Aminco-Bowman spectrophotofluorometer, equipped with a thermoelectric cooler to maintain a constant temperature at 25” C, and a Beckman linear recorder for automatic recording of the changes in fluorescence with time. Procedure. DETERMINATION OF PEROIXDASE. To 2.7 ml of 0.1M tris buffer, pH 8.50, is added 0.1 ml of a 0 . 3 z peroxide solution, 0.1 ml of a 2.5 mg per ml solution of homovanillic acid and 0.1 ml of a solution of the peroxidase to be assayed (containing 0.018 to 3 units). The rate of change in the fluorescence of the solution (AFlminute) is re-
corded at a A,, of 315 mp, and a A,, of 425 mp. From calibration plots of AFlminute us. the peroxidase concentration, the amount of this enzyme present can be determined. DISCUSSION
The homovanillic acid solutions appear very stable in aqueous solution, and have been used for weeks with no appreciable fluorescence formed (hF = 0 after 4 weeks at room temperature in daylight). More data on the comparative stabilities of the reagents in these two methods will be published in a future article. The variation of the rate of oxidation of homovanillic acid, expressed as the rate of formation of the fluorescent oxidized product (11) with time (AFlminute), with changes in the peroxide and peroxidase concentration are indicated in Table I. Using this method, as little as lo-” mole/ml of peroxide and unit/ml of peroxidase are determinable. Full details on the use of homovanillic acid as a fluorometric substrate for the determination of peroxide and peroxidase, as well as other oxidases and their substrates, will appear in a future article. GEORGE G. GUILBAULT Dept. of Chemistry Louisiana State University in New Orleans New Orleans, La.
DAVID N. KRAMER
(1) D. F. Sharman, Brit. J. I’harmacol., 20,204 (1963).
( 2 ) T. Sato, J. Lab. Clin. Me& 66, 517 (1965). (3) H. Corrodi and B. Werdinius, Acta Chem. Scand., 19, 1854 (1965). (4) A. S. Keston and R. Brandt, Anal. Biochem., 11, 1, 6 (1965). (5) W. A. Andreae, Nature, 3.75,859 (1955). (6) A. C. Maehly and B. Chance, “Methods of Biochemical Analysis,” D. Glick, ed., Vol, I, p. 357, Interscience, New York, 1954.
ETHELHACKLEY
Defensive Research Dept. Research Labs Edgewood Arsenal, Md. RECEIVED for review, August 17, 1966. Accepted December 14, 1966. VOL 39, NO. 2, FEBRUARY 1967
271
