Proton hyperfine splitting in the ESR spectra of a stable hydroxynitroxide and its esters application to an ESR assay procedure for alkaline phosphatase

332

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 26777

PROTON H Y P E R F I N E S P L I T T I N G IN T H E E S R SPECTRA OF A STABLE H Y D R O X Y N I T R O X I D E AND ITS ESTERS A P P L I C A T I O N TO AN ESR ASSAY P R O C E D U R E FOR A L K A L I N E PHOSPHATASE

P A U L MUSHAK, J U N E S. TAYLOR AND J O S E P H E. COLEMAN

The Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Conn. 065±0 (U.S.A.) (Received August 23rd, 1971)

SUMMARY

A pronounced difference in resolution of the proton superhyperfine splitting of the ESR signals of the nitroxide 2,2,6,6-tetramethyl-4-piperidinol-I-oxyl (I) and its phosphate (ll) and acetate (III) esters is observed at millimolar concentrations of the nitroxides in aqueous solution at pH 8. This is ascribed to the fact that the charge on the ester group acts as a barrier to the close approach of the molecules and thus leads to a decreased rate of electron spin exchange for the charged species. The g values of this series of nitroxides are observed to vary in the order I I > I I I > I. Radical I I is a substrate for alkaline phosphatase, from E s c h e r i c h i a coli, while radical I is one of the cleavage products. A semiquantitative ESR assay of alkaline phosphatase activity is described, in which the observable parameters are the loss of proton splitting and the shift in g value as the substrate (II) is hydrolyzed to product (I).

INTRODUCTION

The electron-proton hyperfine coupling constants have been determined for a number of stable piperidine nitroxide radicals by BRIERE et al. 1 and KREILICK2. The magnitudes of the proton coupling constants depend on the conformation of the piperidine ring1, ~. These workers found proton hyperfine coupling constants ranging from 0.2 to 0.4 Gauss for the methyl protons and 0.3 to 0.4 Gauss for the r-methylene protons. The proton coupling constants are thus only a few percent of the nitrogen splitting. The proton hyperfine splitting is not observed in the ESR spectrum except at low power in deoxygenated solutions. In the course of an investigation using spin-labeled phosphate monoesters to probe structure-function relationships in alkaline phosphatase, we encountered variBiochim. Biophys. Acta, 261 (1972) 332-338

ESR oF NITROXIDE ESTERS

333

able resolution of the proton hyperfine splitting in the ESR spectra of the hydroxy nitroxide (I) and its phosphate (II) and acetate (III) esters. C~ CH 3

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All three radicals show proton splitting of about 0.4 Gauss. However, the degree of resolution of the proton hyperfine structure observed in aqueous solution at millimolar radical concentrations depends on the charge at the 4-position. This characteristic difference proved useful in monitoring the extent of the conversion of II to I by alkaline phosphatase. MATERIALS AND METHODS

Materials Phosphate ester, label II, was prepared and purified by the published procedure (ref. 3). Nitroxides I and III were obtained by methods similar to those previously employed 4. Enzyme solution Escherichia coli alkaline phosphatase was obtained and purified by methods reported elsewhereS, e. Enzyme solutions were made up in o.oi M Tris buffer (pH 8.0). Preparation of label solutions Nitroxide solutions of varying concentrations (o.o8-2.6 raM) were made up in o.oi M Tris buffer, (pH 8.0) or in heptane. Solid samples of II over a period of time appear to contain reduced label which necessitates storage of prepared solutions overnight with exposure to the atmosphere to achieve a maximum resonance signal. Owing to the relatively low solubility of phosphate ester II in heptane, only nitroxides I and III were studied in this solvent. Methods Preliminary studies of nitroxides. Solutions of the nitroxides were &gassed just prior to spectral measurement by passing a moderate stream of N~ through the solutions for about I rain. (Longer degassing times gave no improvement in resolution.) An open capillary sample tube was used; no loss of resolution occured over the times required for these experiments. Alkaline phosphatase assay procedure. Varying amounts of the enzyme in o.oi M Tris buffer (pH 8.0) were added to 2.6 mM solutions of the ester (II) to yield final enzyme concentrations of o.oi-3.7/~M. Solutions were quickly degassed as above and the ESR spectra recorded. Hydrolysis was monitored by loss of the superhyperfine structure in the spectra as well as by the upfield shift of the signal, the latter reflecting a change in g value (see below). Concomitant assay of inorganic phosphate released on hydrolysis of II was carried out by the method of AMESL ESR measurements. All spectra were obtained with a Varian Model E-4 ESR Biochim. Biophys. Acta, 261 (1972) 3 3 2 - 3 3 8

P. MUSHAKet al.

334

spectrometer using a IOO kHz modulation frequency and an irradiation frequency of 9.I5 GHz. Spectra are presented as the first derivative of the absorption curve. Crystalline diphenyl-picrylhydrazyl was used for field calibration, taking g ---- 2.0036. Unless otherwise stated, spectra were taken with power at 0.85 m W and a modulation amplitude of o.125 Gauss. RESULTS AND DISCUSSION

The effects of radical concentration, charge, and solvent viscosity on the E S R spectrum The ESR spectra of degassed aqueous solutions of radicals I, I I and I I I at 80#M are shown in Fig. I. Power levels less than 2 m W and low modulation amplitudes are necessary to avoid broadening the proton hyperfine structure. Under these conditions,

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Fig. I. The ESR spectra of hydroxynitroxide radical I (A) and its acetate (13) and phosphate (C) esters. The solutions contained 80 #M radical, o.oi M Tris-HC1 buffer (pH 8). Modulation amplitude, 0.2 Gauss; power, 1.85 roW; temp., 20°. all three radicals display proton hyperfine splitting due to the methyl and methylene protons1, 7. At least eight equally spaced lines can be seen on the two downfield peaks (about 323 ° and 3248 Gauss). As the nitroxide concentration is increased, differences in resolution of the proton splitting appear. At 0.5 raM, proton splitting in the spectrum of the alcohol (Fig. 2A, 2) is clearly less well resolved than in the acetate ester spectrum (Fig. 2B, I). At 2.6 mM, the proton splitting has disappeared from the spectra of the alcohol and acetate ester, but can still be observed in the phosphate ester spectrum (Fig. 3). This loss of proton hypertine structure around millimolar concentrations m a y be ascribed to the effect of electron spin exchange 8. The efficiency of spin exchange as a relaxation mechanism increases with the concentration of the paramagnetic species

Biochim. Biophys. Acta, 261 (1972) 332-338

E S R oF NITROXlDE ESTERS

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3234 GAUSS 2 GAUSS F i g . 2. E S R s p e c t r a o f n i t r o x i d e r a d i c a l s I a n d I I I a t 0 . 5 m M i n h e p t a n e a n d w a t e r . A . T h e c e n t e r p e a k of t h e n i t r o x i d e s p e c t r u m o f r a d i c a l I i n h e p t a n e (I) a n d i n o . o i M T r i s - H C 1 b u f f e r ( p H 8) (2). B . T h e c e n t e r p e a k o f t h e s p e c t r u m o f r a d i c a l I I I i n h e p t a n e (2) a n d i n o . o i M T r i s - H C 1 b u f f e r ( p H 8) ( i ) . T e m p . , 20 °.

a n d causes b r o a d e n i n g a n d e v e n t u a l collapse of nuclear h y p e r i i n e s t r u c t u r e as t h e c o n c e n t r a t i o n is increased. T h e m e c h a n i s m of spin exchange d e p e n d s on close app r o a c h of t h e p a r a m a g n e t i c molecules, a n d its efficiency is i n v e r s e l y p r o p o r t i o n a l to ~//T, t h e r a t i o of solvent viscosity to absolute t e m p e r a t u r e s. I n h e p t a n e a t 20 °, ~/T is 4 1 % of t h a t for w a t e r a t 20 °, a n d we observe no p r o t o n s p l i t t i n g for either n i t r o x i d e Biochim.

Biophys.

Acta,

261 (1972) 3 3 2 - 3 3 8

P. MUSHAK et al.

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:Fig. 4. The effect of p H on the ESR spectrum of radical II. The center peak of the spectrum of radical II, 0.5 mM in o.oi M Tris buffer, is shown at pH 1.5, 4.0 and 8.0. Temp., 25 °. I o r I I I a t o. 5 m M (Figs. 2 A a n d 2 B ) . ( R a d i c a l I I is t o o i n s o l u b l e i n h e p t a n e t o p e r m i t o b s e r v a t i o n . ) I f t h e a q u e o u s s o l u t i o n s of Fig. 2 a r e o b s e r v e d a t 60 °, w h e r e BIT is e q u i v a l e n t t o t h a t of h e p t a n e a t 20 °, t h e p r o t o n s p l i t t i n g is also u n d e t e c t a b l e , as e x p e c t e d (not shown). I n a s m u c h as t h e c h a r g e o n t h e r a d i c a l a p p e a r e d t o b e t h e d e t e r m i n i n g f a c t o r i n

Biochim. Biophys. Acta, 261 (1972) 332-338

E S R on NITROXIDE ESTERS

337

the efficiency of spin exchange, it was of interest to observe the effect on the ESR spectra of varying the charge on the phosphate group. ESR spectra for nitroxide I I at p H 1. 5, 4, and 8 are shown in Fig. 4. The resolution of the proton splitting decreases with the charge on the phosphate. A solution of I displayed no change over the same p H range, demonstrating that the change is due to the negative charge on the phosphate group rather than to proton interactions at the nitroxide group. The three nitroxides show slight differences in g values, shown by the shift of the center peak along the field axis (Fig. 3). Using crystalline diphenylpicrylhydrazyl as a reference, the g values estimated for the three radicals in o.oi M Tris-HC1 buffer (pH 8) are: I, g -- 2.0056; I I , g = 2.0060; I I I , g - - 2 . 0 0 5 8 . BRIERE et al. ~ report a g value of 2.0055 for I. E S R assay of alkaline phosphatase The differences between the E S R absorption of the alcohol (I) and the phosphate (II) radicals, though small, are sufficient to be used for a novel, semi-quantitative assay of alkaline phosphatase activity. Buffered aqueous solutions of phosphate ester I I at 2.6 mM were treated with varying amounts of the enzyme, from o.oi #M to 3.7 #M, degassed for I min, and the ESR spectra recorded at periodic intervals between l = I rain and t = I h. Optimal enzyme concentration for this procedure is about 2 #M. Typical results at 2 #M enzyme are shown in Fig. 5. The extent of hydrolysis of I I was checked independently b y measuring the inorganic phosphate formed. Immediately after measuring the E S R spectrum, the reaction was stopped by diluting with acid, and the samples were assayed for inorganic phosphate. Mixtures of I and I I were prepared with percentages of I corresponding to

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F i g . 5. T h e e f f e c t o f e n z y m i c h y d r o l y s i s o n t h e E S R s p e c t r u m o f r a d i c a l I I . S o l u t i o n c o n t a i n e d i n i t i a l l y 1.8 # M a l k a l i n e p h o s p h a t a s e , 2 . 6 m M r a d i c a l I , a n d o . o i M T r i s - H C 1 b u f f e r ( p H 8). T h e c u r v e s l a b e l e d 1 - 7 c o r r e s p o n d t o 2, i i , 21, 36, 61, 9 1 , a n d 121 m i n o f r e a c t i o n t i m e , r e s p e c t i v e l y . T e m p . 25 ° .

Biochim. Biophys. Acta,

261 (1972) 3 3 2 - 3 3 8

338

P. MUSHAKet al.

the amount of inorganic phosphate found. ESR spectra of the mixtures simulated very well the spectra obtained from the corresponding enzyme hydrolysis solution. The extent of the reaction can be observed qualitatively by observing the disappearance of the proton splitting, since at radical concentrations used the alcohol I has no observable proton splitting. I t is also possible to make an estimate of the extent of hydrolysis by measuring the upfield shift as the reaction proceeds. (The capillary sample tube must be left in place in the ESR cavity for the duration of the measurements, otherwise the shifts resulting from variation in alignment of the tube in the cavity may obscure the very small shift in the g value between substrate and product.) Calculations based on initial rates during the first few minutes of hydrolysis yield a hydrolysis rate of 19o #moles I I hydrolyzed per h per mg enzyme, in o.oi M Tris-HC1 buffer, (pH 8), 25 °. This is similar to the rate reported for the hydrolysis of p-nitrophenyl phosphate by alkaline phosphatase under the similar conditions. ACKNOWLEDGMENTS

This work was supported by Grant BO-I3344 from the National Science Foundation and by Grant AM-o9o7o-o7 from the National Institutes of Health, U.S. Public Health Service. REFERENCES I R. BRIERE, H. LEMAIRE, A. RASSAT, P. REY AND A. ROUSSEAU, Bull. Soc. Chim. France,

(1967) 4479. R. W. KREILIC:<,J. Phys. Chem., 46 (1967) 4260. G. C. K. ROBERTS,J. HANNAHAND O. JARDETZK¥,Science, 165 (1969) 504. R. BRIERE,H. LEMAIREAND A. RASSAT, Bull. Soc. Chim. France, (1965) 3273. M. I. HARRISAND J. E. COLEMAN, J. Biol. Chem., 243 (1968) 5063. M. L. APPLEBURYAND J. E. COLEMAN,J. Biol. Chem., 244 (1969) 308. B. N. AMES,Methods Enzymol., 8 (1966) 115. 8 W. PLACHYAND D. KIVELSON,J. Chem. Phys., 47 (1967) 3312.

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