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Fluorescence methods are described for the assay of the oxidative enzymes-peroxidase, glucose oxidase, and xanthine oxidase-based on the conversion of the nonfluorescent homovanillic acid to the highly fluorescent 2,2'-dlhydroxy-3,3'-dimethoxybiphenyl-5,5'-diacetic acid. The initial rate of formation of this fluorescent compound is measured and related to the ac-Recently, Guilbault, Kramer, and Hackley described the use of homovanillic acid (HVA) for the quantitative determination of peroxidase ( 14). The compound (I) is nonfluorescent, but is converted upon oxidation to the highly fluorescent compound (II), which has a Xex of 315 µ and a Xem at 425 µ. It was the intent of this study to develop fluorometric methods for a variety of oxidative enzyme systems using homovanillic acid, and using these new procedures, to develop specific and sensitive analytical methods

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The body of literature on selective ion electrodes has become very large within the last few years. An excellent literature survey reasonably complete through July 1968 was published recently (7). A survey of the field of ion selective electrodes was provided by Rechnitz (2).The theory and application of anion selective membrane electrodes was published by Pungor (3). Highly useful and selective electrodes for I~, Cl-, and Brhave been developed by the use of a precipitate impregnated membrane electrode using silicone rubber as the inert matrix (4-9).

THE OXIDATION OF GUAIACOL, SEROTONIN, 0-CRESOL., p-CRESOL, m-CRESOL, HOMOVANILLIC ACID, AND m-TYROSINE BY HORSERADISH PEROXIDASE AND HYDROGEN PZROXIDE KEY WORDS: G u a i a c o l , S e r o t o n i n , o -C r e s o l , 1, -t . i c i ,~l , m-Cresol, Homovanillic Acid, m-Tyrosine , I1 rscraJisl! P e r o x i d a s e , Hydrogen P e r o x i d e . P a u l J. R r i g n a c , J r . and Tien-hua Yu Department of Chemistry arid L'hys i c s Northwestern S t a t e U r i i v e r s i t i o t Imuisiand N a t c h i t o c h e s , L o u i s i a n a 7145 7 ABSTRACT A s u r v e y of 14 s u b s t r a t e s f o r t h e f l i i o r o m e t r i c d e t e r m i n a t i o n of t h e o x i d a t i v e enzyme-horseradish p e r o x i d a s e was c a r r i e d o u t . The compounds which seemed t o a c t w e l l as s u b s t r a t e s a r e s e r o t o n i n , g u a i a c o l , o -c r e s o l , p -c r e s o l , m-cresol, h w o v a n i l l i c a c i d and m-tyrosine. L-Epinephrine, e q u i l e n i n , e q u i l i n , 6 -e s t r a d i o l , e s t r i o l , and e s t r o n e were s t u d i e d and found n o t t o be s u b s t r a t e s of t h e system. The k i n e t i c s of t h e o x i d a t i o n r e a c t i o n s werr s t u d i e d . They f o l l o w e d t h e M i c h a e l i s c o n s t a n t r a t e e q u a t i o n . The M i c h a e l i s cons t a n t , Km, was determined by a Lineweaver-Burk p l o t f o r e a c h s u b s t r a t e . I t would a p p e a r t h a t t h e p r e s e n c e of a group i n t h e o r t h o , p a r a , o r meta p o s i t i o n of t h e p h e n o l i c r i n g d e c r e a s e s t h e b i n d i n g o f t h e BRIGNAC AND YU s u b s t r a t e t o t h e enzyme's a c t i v e s i t e , a s shown by t h e Km v a l u e s obtained. cluded t h a t m-tyrosine is t h e b e s t s u b s t r a t e (lowest Km v a l u e ) when compared t o a l l t h e s u b s t r a t e s which have been s t u d i e d t o d a t e by t h e a u t h o r s .

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