New reagents for spectral titration of active site of chymotrypsin

Journal of Btochemtcal and Biophysical Methods, 27 (1993) 261-265

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© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-022X/93/$06.00

JBBM 01021

Short Note

New reagents for spectral titration of active site of chymotrypsin Hyung Soon Park, Sook Lee and J u n g h u n Suh * Department of Chemtstry, Seoul Nattonal Unwersity, Seoul 151-742 (South Korea) (Received 29 March 1993) (Accepted 3 June 1993)

Summary The reaction of a-chymotrypsin (ChT) with 2-phenyl-(E)-[4-(E)-cinnamylidene]oxazolin-5-one(EPCO) or 2-phenyl-(Z)-[4-(E)-cinnamylidene]oxazolin-5-one(Z-PCO) at pH 4.5 and 25°C led to the quantitative accumulation of the corresponding acyl-ChT intermediate. Very large changes in molar extinction coefficient were observed in the visible region during the conversion of the oxazolinones to the acyl-ChT intermediates. This afforded a reliable method for the spectral titration of ChT in the visible region. Both E-PCO and Z-PCO were stable under these conditions and did not cause any complication in the titration of the active site. Key words: Chymotrypsin; Active-site titration; Visible region

Titration of the active site of an enzyme is an important subject in the study of the enzyme [1], and methods for the titration of active sites have been reported for several enzymes [2-8]. The active site of ChT has been titrated by using N-(E)cinnamoylimidazole (CI) at pH 5.0 [2]. Under these conditions, acylation of ChT by CI is much faster than the breakdown of the resulting acyl-ChT, leading to quantitative accumulation of the acyl-enzyme intermediate. From the absorbance changes observed due to the accumulation of the acyl-enzyme intermediate, the concentration of the active site is calculated. This method, however, suffers from some drawbacks such as considerably fast spontaneous hydrolysis of CI and the wavelength (335 nm) used for measurement of absorbance changes. Although satisfactory results are obtained when purified ChT is used, this method is not applicable in the presence of additives that destabilize CI. In addition, this method * Corresponding author.

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E-PCO

Z-PCO Scheme 1.

is not useful when chromophores strongly absorbing in the near UV region are introduced as a part of additives or as the consequence of chemical modification or conjugation to polymeric supports of the enzyme. In this paper, we report new reagents (E-PCO and Z-PCO) for spectral titration of ChT which are considerably more stable to spontaneous hydrolysis than CI and afford spectral titration in the visible region. E-PCO and Z-PCO were prepared as follows. The mixture of hippuric acid (5 g), (E)-cinnamaldehyde (3.7 g), anhydrous sodium acetate (2.3 g), and acetic anhydride (8.5 g) was heated at 100°C for 2 h, and 15 ml ethanol was added slowly to the mixture. Crystalline products (a mixture of Z-PCO (60%) and E-PCO (40%)) formed after the mixture was left overnight at room temperature were separated by filtration and washed with cold ethanol and water. The two isomers of PCO were separated on a silica gel column by eluting with benzene (the Z isomer was eluted first) and each was further purified by recrystallization from benzene. Analytical data for E-PCO: melting point, 162-163.5°C. 1H-NMR (CDC13): 3 7.09 (d, 1 H), 7.28 (d, 1 H), 7.35-7.63 (m, 8 H), 8.05-8.08 (m, 2 H), 8.11 (q, 1 H). Calculated for C18H13NO2: C, 78.53; H, 4.76; N, 5.09. Found: C, 78.95; H, 4.75; N, 5.16. Analytical data for Z-PCO: melting point, 146-147.5°C. 1H-NMR (CDCI3): 3 7.12 (d, 1 H), 7.14 (d, 1 H), 7.33-7.65 (m, 8H), 7.70 (q, 1 H), 8.13-8.16 (m, 2 H). Calculated for C18H13NO2: C, 78.53; H, 4.76; N, 5.09. Found: C, 78.90; H, 4.90; N, 5.12. Structures of the E and Z isomers were assigned on the basis of chemical shifts for H , (3 7.28 for E-PCO and 7.14 for Z-PCO) [9] as well as coupling constants J(CO, H~) (48.6 Hz for E-PCO and 15 Hz for Z-PCO) [10]. It has been reported that ChT makes nucleophilic attack at the carbonyl carbon atom of an 2-phenyloxazolin-5-one [11], opening the heterocyclic ring of the oxazolin-5-one. Breakdown of the resultant acyl-ChT intermediate completes the hydrolysis of the oxazolin-5-one. When E-PCO or Z-PCO was mixed with ChT, accumulation of an intermediate (ES'; Eqn. 1)was observed spectrophotometrically. Intermediate ES' would accu-

263 0.2 a

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Fig. 1. Electronic spectra of E-PCO (la: 3.4× 10-6 M), the acyl-ChT intermediate formed with E-PCO (lb: 3.4× 10-6 M), Z-PCO (2a: 3.1 × 10-6 M), and the acyl-ChT intermediate formed with Z-PCO (2b: 3.1 x 10-6 M). mulate in a quantitative amount when the rate of the formation step of the intermediate is much faster than that of the corresponding breakdown step. E+S ~ ES ~ ES'-~ E+P,

(1)

The electronic spectra of E - P C O and Z - P C O and those of the corresponding acyl-ChT intermediates are illustrated in Fig. 1. Differences in absorbances at 350-440 nm between the titrating reagents and the respective acyl-ChT intermediates allow spectral titration of ChT in the visible region. The spectral titration was performed at p H 4.5 and 25°C in the presence of 0.5 M NaCI, 0.02 M sodium acetate and 8.2% ( v / v ) dimethyl sulfoxide. ChT (EC 3.4.21.1) was purchased from Sigma (St. Louis, MO, USA). Stock solutions of E - P C O and Z - P C O were p r e p a r e d in dimethyl sulfoxide. When ChT was added to the buffer solution containing E - P C O or Z-PCO, accumulation of E S ' completed within 1 - 2 min. Spontaneous hydrolysis of E - P C O or Z - P C O and breakdown of ES' was not appreciable under these conditions. The absorbance changes (AAbs) accompanying the accumulation of ES' may be measured by varying S O (the initially added concentration of the substrate) with E 0 (the initially added concentration of ChT) fixed at a constant value (Method A) as exemplified by Fig. 2. Alternatively, AAbs can be measured by varying E 0 with S O fixed at a constant value (Method B) as illustrated in Fig. 3. The intersecting point of the two straight lines (Fig. 2 or 3) represents the equivalence point of the active-site titration.

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S O(10 -~ M) Fig. 2. The plot of - AAbs (at 400 nm) against SO with E o fixed at 2.1 x 10 -6 M (as determined by titration with CI) in the active-site titration of ChT with E-PCO.

T h e s l o p e o f t h e s t r a i g h t line s e e n b e l o w t h e e q u i v a l e n c e p o i n t (Fig. 2 o r 3) c o r r e s p o n d s to t h e d i f f e r e n c e ( A e = e s - e E S ) in t h e m o l a r e x t i n c t i o n c o e f f i c i e n t s b e t w e e n t h e s u b s t r a t e a n d t h e a c y l - C h T . T h e v a l u e o f Ae f o r E - P C O at 400 n m was e s t i m a t e d as - 24 900 c m - t M - ~ by M e t h o d A o r - 24 800 c m - ~ M - l by M e t h o d B. T h a t o f Z - P C O at 400 n m was e s t i m a t e d as - 2 3 6 0 0 c m - l M -~ by M e t h o d A o r - 23 000 c m - ~ M - ~ by M e t h o d B. T h e c o n c e n t r a t i o n o f C h T c a n b e c o r r e c t l y m e a s u r e d by c o n s t r u c t i n g t i t r a t i o n d i a g r a m s s u c h as t h o s e i l l u s t r a t e d in Fig. 2 o r 3. I n s t e a d , m o r e c o n v e n i e n t , a l t h o u g h less p r e c i s e , q u a n t i t a t i o n c a n b e

,

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Eo (lO-" M) Fig. 3. The plot of - / t A b s (at 400 nm) against E 0 with So fixed at 3.2x10 -6 M in the active-site titration of ChT with E-PCO.

265 m a d e by simply m e a s u r i n g A A b s with t h e a d d i t i o n o f excess titrant and dividing it with Ae ( E 0 = AAbs/Ae). T h e c o n c e n t r a t i o n o f C h T d e t e r m i n e d by this m e t h o d a g r e e d within a few p e r cents with that e s t i m a t e d by th e c o n v e n t i o n a l m e t h o d b ased on CI. A s m e n t i o n e d above, t h e n ew m e t h o d has several a d v a n t a g e s c o m p a r e d with t h e c o n v e n t i o n a l m e t h o d d u e to the g r e a t e r stability of th e titrating r e a g e n t s an d t h e collection o f spectral data in the visible region.

Acknowledgements This w o r k was s u p p o r t e d by t h e Basic S c ie nce R e s e a r c h I n st i t u t e P r o g r a m o f R . O . K . Ministry o f E d u c a t i o n . H.S.P. thanks t h e D a e w o o F o u n d a t i o n for a p r e d o c t o r a l fellowship.

References 1 Kezdy, F.J. and Kaiser, E.T. (1970) Principles of active site titration of proteolytic enzymes. In: Perlman, G.E. and Lorand, L. (Eds.), Methods in Enzymology, Vol. XIX, Academic Press, New York, pp. 3-20. 2 Schonbaum, G.R., Zerner, B. and Bender, M.L. (1961) The spectrophotometric determination of the operational normality of an a-chymotrypsin solution, J. Biol. Chem. 236, 2930-2935. 3 Bender, M.L. (1971) Mechanisms of homogeneous catalysis from protons to proteins, Wiley-Interscience, New York, pp. 397-455. 4 Mandez, W.M., Jr. and Kaiser, E.T. (1975) A reverse burst active site titration procedure for human carbonic anhydrase B, Biochem. Biophys. Res. Commun. 66, 949-955. 5 Fersht, A.R. (Ed.) (1977) Enzyme structure and mechanism, Freeman, San Francisco, pp. 122-126. 6 Bechet, J.J., Jouadjeto, M. and d'Albis, D. (1986) Active-site titration of enzymes at high concentration. Application to Myosin ATPase, Eur. J. Biochem. 161,343-349. 7 Roth, M. and Selz, L. (1988) Molecular titration as a means of calibrating enzyme reference materials, Clin. Chim. Acta 173, 27-34. 8 Suh, J., Hwang, B.K., Jang, I. and Oh, E. (1991) Spectral titration of active site of carboxypeptidase A, J. Biochem. Biophys. Methods 22, 167-170. 9 Rao, Y.S. and Filler, R. (1975) Geometric isomers of 2-aryl(aralkyl)-4-arylidene(alkylidene)-5(4H)oxazolones, Synthesis 749-764. 10 Letcher, R.M. and Acheson, R.M. (1981) Vicinal C, H spin coupling constants in determining alkene stereochemistry, Org. Mag. Res. 16, 316-318. 11 Hamilton, S.E. and Zerner, B. (1981) Solvent effects on the deacylation of acyl-chymotrypsins: a critical comment on the charge-relay hypothesis, J. Am. Chem. Soc. 103, 1827-1831.