A rapid spectrophotometric method for the determination of esterase activity

Journal of Biochemical and Biophysical Methods, 3 (1980) 345--354 © Elsevier/North-Holland Biomedical Press

345

A RAPID SPECTROPHOTOMETRIC METHOD FOR THE DETERMINATION OF ESTERASE ACTIVITY *

ROBERT B. MILLER ** and ROBERT C. KARN Department of Medical Genetics, Indiana University School of Medicine, 1100 West Michigan Street, Indianapolis, IN 46223, U.S.A.

(Received 5 May 1980; accepted 24 June 1980)

We have developed a spectrophotometric assay method which continuously records esterase activity at 510 nm by monitoring absorbance changes due to the formation of a diazo dye complex. In our method, ~-naphthyl ester substrates are hydrolyzed by enzymatic action to a-naphthol which couples to Fast Blue RR salt (a diazonium salt) forming a diazo dye complex. Our method is unique in directly monitoring the formation of the diazo dye complex without extracting the color of the complex as in other methods that use naphthyl esters and diazo coupling of reaction products. The method appears to be limited to ~-naphthyl ester substrates, however, since/3-naphthyl esters did not give a linear change in absorbance in the enzymatic reactions tested. With this assay method, one can use a single substrate both to determine esterase units quantitatively in solution and to detect esterase staining activity on gel electrophoresis. Key words: esterase; spectrophotometric assay; diazo coupling.

INTRODUCTION Several s p e c t r o p h o t o m e t r i c m e t h o d s have been de~eloped f o r m e a s u r i n g esterase activity [ 1 - - 4 , 6 - - 7 ]. S p e c t r o p h o t o m e t r i c m e t h o d s t h a t use c h r o m o genic substrates measure the a m o u n t o f p r o d u c t f o r m e d b y r e c o r d i n g t h e visible light a b s o r b a n c e o f either the p r o d u c t itself o r a p r o d u c t - - d y e c o m plex [ 1 - - 4 , 6 ] . Liberti [1] d e v e l o p e d a s p e c t r o p h o t o m e t r i c m e t h o d f o r measuring esterase activity using colorless p - n i t r o p h e n y l p r 0 p i o n a t e as substrate. B y e n z y m a t i c a c t i o n , the substrate was h y d r o l y z e d to a c o l o r e d p-nitrop h e n o l p r o d u c t w h i c h was c o n t i n u o u s l y m e a s u r e d at 4 0 5 n m . E l l m a n et al. [2] d e v e l o p e d a s p e c t r o p h o t o m e t r i c m e t h o d f o r measuring acetylcholinesterase activity b y m e a s u r i n g t h e a b s o r b a n c e o f a p r o d u c t - - d y e c o m p l e x . I n t h a t m e t h o d , a c e t y l c h o l i n e s t e r a s e activity was m e a s u r e d b y c o n t i n u o u s l y r e c o r d i n g the a b s o r b a n c e o f a y e l l o w c o l o r e d p r o d u c t at 4 1 2 n m resulting * This is publication No. 79-10 from the Department of Medical Genetics, Indiana University School of Medicine. ** Present address: Department of Pediatrics, East Tennessee State University, College of Medicine, Johnson City, TN 37601, U.S.A.

346 from the reaction of thiocholine (enzyme product) and dithiobisnitrobenzoate. Spectrophotometric esterase determinations employing naphthyl esters as substrates use coupling reactions to form insoluble product--dye complexes. As a result, the color produced by the product--dye complex must be extracted with an organic solvent before recording absorbance measurements at ~sible wavelengths. Several spectrophotometric methods for esterase, using napthyl ester substrates, have been reported [3,4]. The purpose of this paper is to describe a simplified spectrophotometric method for measuring esterase activity using naphthyl esters and diazo coupling of reaction products. Using our method, one can continuously record the absorbance of diazo dye complex formation without extracting the colored complex with organic solvents. In the spectrophotometric esterase assay reported here, the concentration of naphthyl esters and diazonium salt was carefully controlled to prevent precipitation of the diazo dye complex, enabling direct and continuous measurement of diazo dye formation. This improvement makes our esterase assay method more rapid and easier to perform than other methods employing diazo coupling. Additionally, it is possible to compare esterase units determined quantitatively in solution with esterase staining reactions that also employ diazo coupling for locating areas of esterase activity on electrophoresis gels and in tissues. MATERIALS AND METHODS Materials

The a and ~ forms of naphthol, naphthyl acetate, naphthyl propionate, and naphthyl butyrate; Fast Blue R R salt (diazonium salt of 4-benzoylamino-2,5dimethoxy-aniline-ZnCl2); sodium phosphate (mono, di-, and tri-basic); and hog liver carboxylesterase (EC 3.1.1.1) (3.3 mg/ml) were obtained from Sigma Chemical Co. Instruments

Spectrophotometric determinations Recording Spectrophotometer. For recorded at room temperature (25°C), inch/min. A Beckman Model 3500 pH ues of all solutions used in the assays. Me th o ds Reagents.

were made using a Gilford 2400 routine assays, the tracings were at 510 nm, and a chart speed of 0.5 Meter was used to determine pH val-

All substrates were 0.016 M a or fl-naphthyl esters in acetone. Solutions (0.0016 M) of the a and/3 forms of naphthol were also prepared in acetone. Fast Blue R R salt (0.00345 M) was prepared with distilled water and kept on ice. This helped maintain stability for at least 2 h. Sodium phosphate buffers (0.1 M) were prepared at 0.5 pH intervals from pH 6.0 to 9.5. A 1 : 800 dilution of commercial hog liver carboxylesterase in water or

347 buffer was used to establish the assay conditions. Procedures. The development of the spectrophotometric assay involved testing several parameters: (a) the absorbance spectra between 370 nm and 660 nm; (b) the effect of pH on product formation at 510 nm; (c) the effect of pH on the molar absorption of the diazo dye complex; and (d) the relationship between product concentration and absorbance. For a routine assay using hog liver carboxylesterase, the following order of additions was made into a 1.0 ml cuvette: sodium phosphate buffer, enzyme, Fast Blue RR salt and naphthyl ester substrate. A unit of activity was defined as the a m o u n t of enzyme that hydrolyzed 1.0 pmole of substrate per minute at 25°C. For calculating units, units =A/min/e X IX 106, where A / m i n = the initial slope of absorbance change, e = molar absorption coefficient, and l = 1.0 cm light path length. Using routine assay conditions, the effect of enzyme concentration and the pH optimum of hog liver carboxylesterase was examined. Variations in the assay methods are described in the figure legends. For applying our assay method, total human serum esterase activity was measured using normal serum and several a- and ~-naphthyl esters as substrates. RESULTS The absorbance spectrum (Fig. l ) demonstrates maximal absorbance of the a-naphthol--Fast Blue RR diazo complex at 510 nm and minimal absorbance of a-naphthyl acetate in the presence of Fast Blue RR salt at that wavelength. Substituting ~-naphthol for a-naphthol gave the same results with m a x i m u m absorbance of the 13-naphthol--Fast Blue RR diazo complex occurring at 510 nm. For estimating the molar absorption coefficient, the following equation was used: e = A/c X l, where A = the absorbance of the a naphthol in the reaction mixture, and l = 1.0 cm light path length. The molar absorption coefficient determined was 1.9 • 1071 • mo1-1 • cm -1. Diazo coupling reactions involving diazonium salts, such as Fast Blue RR Salt, and naphthols are strongly influenced by the pH of the coupling medium [5]. The effect of pH on coupling a-napthol t o Fast .Blue RR salt is presented (Fig. 2). At pH values below pH 7.5, the coupling reaction is slow because the h y d r o x y l group in 1-position on ~-naphthol is in the acidic form (-OH) reducing the rate of coupling. At pH values of pH 7.5 and above, the coupling reaction is essentially instantaneous because the h y d r o x y l group is ionized (-O-) and powerfully electron releasing. When measuring esterase acti~.'ty at pH 7.5 or greater, the enzymatic reaction is the rate-limiting step. The molar absorption of the product diazo complex (a-naphthol--Fast Blue RR complex) was also affected by pH (Fig. 3). After forming the diazo complex at pH 7.5, aliquots of the diazo complex were diluted into buffers at pH 6.0, 7.0, 8.0 and 9.0. The results showed a decrease in absorbance at low pH and an increase in absorbance at high pH. The state of ionization of the h y d r o x y l group on a-naphthol appears to affect the absorbance of the

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Fig. 1. A b s o r b a n c e s p e c t r u m . T h e a b s o r b a n c e s p e c t r u m o f ~he s u b s t r a t e ~ - n a p h t h y l acetate, in t h e presence o f Fast Blue R R salt was d e t e r m i n e d using a 1.0 ml s o l u t i o n c o n t a i n ing 0.16 ~ m o l ~ - n a p h t h y l a c e t a t e , 0.67 # t o o l Fast Blue R R salt a n d 0 . 0 7 9 m m o l s o d i u m p h o s p h a t e buffer, pH 8.0 ( . . . . . . ). T h e a b s o r b a n c e s p e c t r u m o f t h e p r o d u c t diazo c o m plex was d e t e r m i n e d using a 1.0 ml s o l u t i o n c o n t a i n i n g 0 . 0 4 8 p m o l a - n a p h t h o l , 0.67 p m o l Fast Blue R R salt a n d 0 . 0 7 7 m m o l s o d i u m p h o s p h a t e buffer, pH 8.0 ( ). T h e r e a c t i o n m i x t u r e s were i n c u b a t e d for 2 rain before o b t a i n i n g t h e a b s o r b a n c e s at intervals o f 10 nm.

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Fig. 2. E f f e c t o f pH o n p r o d u c t fol~nation. A 1.0 ml r e a c t i o n m i x t u r e c o n t a i n i n g 0 . 0 3 2 p m o l ~ - n a p h t h o l , 0.67 p m o l Fast Blue R R salt a n d a 0 . 0 7 8 m m o l s o d i u m p h o s p h a t e b u f f e r was u s e d to t e s t t h e e f f e c t o f p H o n t h e f o r m a t i o n of t h e c~-naphthol--Fast Blue R R diazo c o m p l e x at t h e pH values s h o w n .

diazo dye complex even after coupling to the diazonium salt. The relationship between product concentration and absorbance is shown in Fig. 4. A pH 8.0 sodium phosphate buffer was used to test this relationship to ensure maximum formation of the a-naphthol--Fast'Blue R R diazo complex after a 2 min incubation of the reaction mixture. At low concentrations, there is a linear response between hog liver carboxylesterase concentration and units of activity. This continues up to 0.8 pg of enzyme in the assay mixture. Further additions of enzyme resulted in non-linearity of enzyme activity due to substrate limitation. Kriscb [6] found 8.0 to be the pH o p t i m u m of hog liver carboxylesterase with p-nitrophenyl acetate as substrate in a spectrophotometric assay continously measuring the formation of p-nitrophenol at 405 nm. Using our assay method, the pH o f hog liver carboxylesterase was also found t o be pH 8.0 (Fig. 5). Because the molar absorption of the diazo complex increases with pH (Fig. 3), the decrease in absorbance at pH 8.5 reflects a real decrease in enzymatic activity. At pH values above pH 8.5, rapid non-enzymatic hydrolysis of the substrate and the formation of diazo hydroxides resulted in non-linear spectrophotometric tracings. Therefore, enzyme activity above pH 8.5 could not be reliably recorded. At pH 8.5 and below, non-enzymatic

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Fig. 3. Effect of pH on molar absorption of product complex. To perform this test, 10X solutions of c~-naphthol and Fast Blue RR salt were coupled in the presence of 0.001 M sodium phosphate buffer, pH 7.5. The reaction mixture contained 0.48/lmol a-naphthol and 6.7 pmol Fast Blue RR sait in the presence of 0.077 pmol sodium phosphate, pH 7.5. The coupling reaction proceeded for 2 min and then 0.1 ml of the reaction mixture was added to 0.9 ml of 0.11 M sodium phosphate buffer. This attained concentrations normally found in routine assay conditions. The figure shows final pH and absorbance at 510 niT)..

h y d r o l y s i s o f the substrate was c o m p e n s a t e d f o r b y s u b t r a c t i n g the changes in a b s o r b a n c e per m i n u t e o f t h e r e a c t i o n m i x t u r e w i t h o u t e n z y m e . Using r o u t i n e assay c o n d i t i o n s , t o t a l n o r m a l h u m a n serum esterase activity was m e a s u r e d using ~- and /3-naphthyl esters (Fig. 6). T h e results show linear changes in a b s o r b a n c e using a - n a p h t h y l esters whereas non-linear changes in a b s o r b a n c e were f o u n d using fi-naphthyl esters. When we e x a m ined the rate o f c o u p l i n g o f fi-naphthol to Fast Blue R R salt, we f o u n d coupling to be very slow at pH 7.5 (0.09 A A / m i n ) whereas t h e c o u p l i n g o f a - n a p h t h o l to Fast Blue R R salt is a l m o s t i n s t a n t a n e o u s at t h a t pH (Fig. 2). Burlina et al. [7] n o t e d t h a t azo c o u p l i n g o f fl-naphthol is rate limiting, affecting the r e a c t i o n velocity in an e n z y m a t i c reaction. Thus, our assay m e t h o d is o n l y suitable for use with a - n a p h t h y l esters, esters n o t b e f o r e used

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Fig. 4. Beer's Law plot. T h e r e l a t i o n s h i p b e t w e e n a b s o r b a n e e a n d p r o d u c t c o n c e n t r a t i o n was t e s t e d b y increasing t h e a m o u n t s o f c~-naphthol a d d e d to t h e r e a c t i o n m i x t u r e cont a i n i n g 0.67 ~ m o l Fast Blue R R salt a n d 0 . 0 7 4 m m o l s o d i u m p h o s p h a t e buffer, pH 8.0. Distilled w a t e r was used to m a k e t h e t o t a l v o l u m e 1.0 ml.

to measure esterase activity in a continuously recording spectrophotometric assay. DISCUSSION

Naphthyl esters are the most commonly-used substrates for the detection of esterase activity on gels following electrophoresis and for the histological detection of esterase activity in tissues [8]. We developed the continuous esterase assay reported here to facilitate direct and rapid comparisons with a single substrate between esterase units determined quantitatively in solution with diazo staining reactions used for locating esterase activity on electrophoresis gels or in tissues. The limitation of our assay m e t h o d is in its use with ~-naphthyl esters. As shown in Fig. 6, ~-naphthyl esters gave a linear response with time up to 2.5 min whereas/3-naphthyl esters only approximate a linear response up to 1.5 min with clear non-linear responses over a longer period of time. The reason for nondinearity may be due to differences in the kinetics o f dye coupling. The coupling reaction between ~-naphthol and diazonium salts involves an electrophilic substitution of ~-naphthol at the 2- or 4-position (the activating

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sodium phosphate buffer at the pH values shown, 0.8 pg hog liver carboxylesterase (diluted I : 800 in distilled water), 0.67 pmol Fast Blue RR salt and 0.56 #tool ~-naphthyl acetate.

hydroxyl group is in the 1- position) [9]. However, the coupling reaction between fl-naphthol and diazonium salts occurs exclusively at the 1- position of .B-naphthol by electrophilic substitution (the activating hydroxyl group is in the 2- position) [9]. Thus, the presence of only one substitution site on ~-naphthoi may be responsible for its reduced rate of coupling with diazonium salts as compared to coupling rates of a-naphthol and diazonium salts [9]. Steric hindrence may also be involved in reducing reaction rates. We are aware of only one spectrophotometric assay that continuously measures the formation of naphthol. That method, developed by Burlina et al. [7], measures the formation of ~.-naphthol (enzyme product) without

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TIME {MIN] Fig. 6. Quantitative esterase assay of h u m a n serum using various n a p h t h y l ester substrates. For applying the assay m e t h o d , six naphthyl ester substrates were used to measure total serum esterase activity. 0.48 ~ m o l naphthyl esters were added to the reaction m i x t u r e containing 0.67 p m o l Fast Blue R R salt, 0.075 m m o l s o d i u m phosphate, pH 7.5, and 20.0 ALl fresh h u m a n serum. The figure shows actual s p e c t r o p h o t o m e t r i c tracings. Because of non-linearity of the tracings with ~-naphthyl esters, units of activity were only d e t e r m i n e d using (~-naphthyl esters (~-naphthyl acetate: 0.0038 units; c~-naphthyl propionate: 0.00857 ; (x-naphthyl butyrate: 0.00536 units).

diazo coupling at 313 nm. The disadvm~tage of that assay is that it measures the formation of product at a wavelength where a variety of naturally occurring biological compounds also absorb. Our assay method obviates that problem because it measures the reaction much higher on the spectrum. Although the observed nonlinearity may limit the use of the assay with fl-naphthyl esters, the assay is clearly useful for measuring esterase activity with a-naphthyl esters, a class of substrates widely used in esterase research. S I M P L I F I E D D E S C R I P T I O N OF T H E M E T H O D AND ITS A P P L I C A T I O N S The s p e e t r o p h o t o m e t r i c m e t h o d described is a rapid and reliable m e t h o d for the determination of esterase activity. ~-Naphthyl ester substrates are h y d r o l y z e d by e n z y m a t i c action to ~-naphthols which couple to Fast Blue R R salt forming a diazo complex. The f o r m a t i o n of the diazo c o m p l e x is m o n i t o r e d directly at 510 n m w i t h o u t extracting the color of the c o m p l e x as in o t h e r m e t h o d s that use ~-naphthyl esters and diazo coupling of

354 reaction products. Our assay method can be used to measure esterase activity from a variety of sources and can be used to compare esterase units determined quantitatively in so!ution with esterase staining activity on gel electrophoresis. ACKNOWLEDGEMENTS

This work was supported in part by The Indiana University Human Genetics Center, PHS 50 GM 21054. R.B.M. was supported by Dental Genetics Training Grant No. PHS T01 DE 011915, and R.C.K. was supported by PHS Career Development Award No. 1 K04 AM 0028403. REFERENCES 1 Liberti, J.P. (1968) Anal. Biochem. 23, 53--59 2 Ellman, O.L., Courtney, K.D., Andres, V. Jr. and Featherstone, R.M. (1961) Biochem. Pharmaco}. 7, 88--95 3 Seligman, A.M. and Nachlas, M.M. (1950) J. CIin. Invest. 29, 31--36 4 Koeppen, A.H. (1969) Clin. Chim. Acta 26, 71--76 5 Zollinger, H. (1961) Azo and Diazo Chemistry, pp. 221--231, Interscience, New York 6 Krisch, K. (1966) Biochim. Biophys. Acta !22, 2 5 6 - 2 8 0 7 Burlina, A. and Galzigna, L. (1972) Clin. Chim. Acta 39, 255--257 8 Augustinsson, K.B. (1968) Biochim. Biophys. Acta 159, 197--200 9 Coffey, S., ed. (1978) Rodd's Chemistry of Carbon Compounds, Vol. iiI, Aromatic Compounds, Part G, 2nd edn., pp. 197, 211. Elsevier Scientific Publ. New York