13C NMR analysis of methionine sulfoxide in protein

Journal o f Biochemical and Biophysical Methods, I (1979) 145--151 © Elsevier/North-Holland Biomedical Press

145

13C N M R A N A L Y S I S O F M E T H I O N I N E S U L F O X I D E I N P R O T E I N

JACK S. COHEN *, JOSEPH YARIV, A. JOSEPH KALB (GILBOA), LEV JACOBSON and YORAM SHECHTER Department o f Biophysics, Isotope Research and Organic Chemistry, The Weizmann Institute o f Science, Rehovot (Israel)

(Received 12 December 1978; accepted 15 March 1979)

The 13ce NMR signal of methionine sulfoxide is 22.6 ppm downfield from that of methionine. This affords a method by which the extent of methionine oxidation can be determined in intact protein. We demonstrate the utility of this approach with ~-galactosidase enriched with 13C in its methionine methyls. Key words: 13C NMR; methionine sulfoxide ; oxidation; ~-galactosidase.

INTRODUCTION M e t h i o n i n e s u l f o x i d e is n o t n o r m a l l y f o u n d in proteins, b u t is o f t e n a p r o d u c t o f their o x i d a t i o n . Analysis o f m e t h i o n i n e s u l f o x i d e has p r e v i o u s l y b e e n carried o u t b y c h r o m a t o g r a p h y o f digests f o l l o w i n g r e d u c t i o n [1]. Here we describe a m e t h o d b y w h i c h t h e ratio o f m e t h i o n i n e s u l f o x i d e to m e t h i o n i n e in a p r o t e i n m a y be d e t e r m i n e d n o n - d e s t r u c t i v e l y and r e p e a t e d l y o n a sample u n d e r g o i n g progressive o x i d a t i o n . This ratio can be o b t a i n e d f r o m t h e i n t e g r a t e d intensities o f t h e '3Ce r e s o n a n c e a b s o r p t i o n s o f the t w o c o m p o n e n t s , which are separated b y 22.6 p p m d u e to t h e de-shielding e f f e c t o f the o x y g e n a t o m . T h e large n u m b e r o f alkyl c a r b o n r e s o n a n c e s and t h e limited sensitivity at natural a b u n d a n c e o f '3C [ 2 ] , necessitates e n r i c h m e n t o f t h e C e m e t h i o nine a t o m s [ 3 - - 5 ] . This was a c c o m p l i s h e d in t h e case of/3-galactosidase b y b i o s y n t h e s i s using a methionine-less m u t a n t o f E s c h e r i c h i a coli. O x i d a t i o n o f the e n z y m e was carried o u t b y a d d i t i o n o f c h l o r a m i n e T, w h i c h has been s h o w n to be a selective r e a g e n t f o r the o x i d a t i o n o f m e t h i o n i n e t o m e t h i o nine s u l f o x i d e [ 1 ] .

* Permanent address: National Institute of Child ttealth and Human Development, N.I.H., Bethesda, MD 20014, U.S.A.

146 EXPERIMENTAL PROCEDUF~ES

3C NMR spectroscopy "3C NMR spectra were measured at 67.88 Mttz with a Broker WH270 spectrometer with a 10 mm pr obe and equipped with a Nicolet 1180 cornouter. Usual operating conditions were: 70 ° pulse (16 ps), 0.45 s acquisition time (9 kHz sweep width), 8000 points (2.2 Hz/cm), exponential line broadening (10 Hz), alternating phase and quadrature detection. Broad band noise-modulated p r o t o n dccoupling power (5 W) centered in the aliphatic p r o t o n region was employed. The deuterium of internal 2H20 (10%) was used for the lock signal. The sample was kept at 20 e 1°C with a flow of cold nitrogen gas. Sample t e m p e r a t u r e was determined by quickly removing the tube from the probe and dipping a glass-coated t h e r m o c o u p l e into the solution. 30 000--80 000 scans were accumulated. Peak areas were determined by the computer's integration subroutine. The relaxation times for all enriched carbon atoms were estimated to be shorter than the interval between pulses by at least a factor of 5, assuring proport i onal i t y between integrated areas and concentration. Chemical shift values are quot ed downfield from external TMS (approx. 50% in CHC13) in a concentric capillary inserted into water.

Growth o f bacteria Two strains of K coIi K-12 were used, strain 3300, constitutive in ~-galactosidase, and an inducible methionine-requiring strain, CS-8. Strain 3300 was grown in a mineral medium supplemented with thiamine and casamino acids as described [6]. Strain CS-8 was grown in a volume of 50 1 from a starter of 4 1. Glycerol/salts medium was supplemented with 25 ~g/1 of ~3C-enriehed L-methionine [7]. Cells were hmwested in the late logarithmic phase. The yield was 96 g o f packed cells. ~3C-enriched methionine was synthesized from S-benzyl-L-homocysteine and [~3C]methyliodide essentially as described by du Vigneaud et al. [8] e x cep t th at S - b e n z y l h o m o e y s t e i n e was used instead of homocystine. 2.25 g o f S-benzyl-L-homocysteine h e m i h y d r a t e (Cyclo Chemicals, Los Angeles, Calif.) was treated with 2 g of m e t hyl i odi d e (90.3% ~3C, Merck, Sharp and Dohme, Canada Ltd.) and the product, in acidified aqueous solution, was extracted with ethylacetate to remove excess reagent. Amino acid analysis established that the p r o d u c t was pure methionine. ~3C NMR spectroscopy confirmed that the pr oduct was enriched solely in the m et hyl carbon. The p r o d u c t was used w i t h o u t further purification to supplement the growth medium of the methionine-requiring strain.

Preparation o f enzyme En zy me, b o th isotopically normal and ~3C-enriched, was prepared as described by Craven et al. [6] with the following i m p o r t a n t changes in

147 procedure: (a) m e r c a p t o e t h a n o l was totally excluded during disruption of bacteria and all subsequent isolation steps; (b) the extract and the redissolved a m m o n i u m sulfate precipitate were spun in r o t o r 35 of a Spinco ultracentrifuge operating at 32 000 rev./min for 1 h to remove sedimentable matter; (c) fractionation on Sephadex G-200 was omitted; (d) for concentration o f en zy me only ultrafiltration was used and not a m m o n i u m sulfate precipitation; (e) isolated protein was stored frozen at --20 ° C. The isolated protein in 0.1 M sodium phosphate, pH 7.0, and 0.01 M MgC12 was characterized by A'~sC~m/A26o~m of 1.9. In an ultracentrifuge, operating at 30 000 rev./min and analyzed with a cell scanner at 280 nm, enriched e n z y m e sedimented with S°0o of 15.2 s with no evidence of impurities. Normal e n z y m e sedimented with S°0o of 14.8 s and contained a small a m o u n t (<5%) of a low-molecular-weight impurity. Specific activities were 690 p m o l / m i n / m g for enriched e n z y m e and 450 pmol/min/mg for normal en zy me.

Oxidation procedure Oxidation of 13C-enriched ~-galactosidase was carried o u t at room temperature (approx. 22°C) by adding aliquots of chloramine T with efficient mixing to e n z y m e in the NMR tube. E n z y m e c o n c e n t r a t i o n at the start of the NMR e x p e r i m e n t was 17.4 mg/ A1% ml (based on 2~.lcm,2SOl m = 20.9) in 0.1 M sodium phosphate, pH 7.0, conraining 0.01 M magnesium chloride and 10% :H20). Chloramine T was added f r o m a 0.1 M stock solution in H~O which was normalized by iodometric titration. Methionine c o n t e n t in the sample was calculated on the basis of 23 meth io n in e residues per subunit of 116, 349 daltons [9] and corresponded to 3.47 pm ol before the first addition of chloramine T. RESULTS AND DISCUSSION The '3C NMR spectra of methionine and m et hi oni ne sulfoxide each contain four signals (Table 1). In the assignment of the resonances of methionine sulfoxide we were guided by t he large and almost equal downfield shifts o f the resonances of carbon atoms adjacent to the sulfoxide group, and a small upfield shift of the next-nearest-neighbor carbon atom resonances, previously observed in penicillins and related sulfoxides [10]. Thus, oxidation of methionine to methionine sulfoxide results in a downfield shift of 22.6 ppm for the m e t hyl carbon resonance. '3C NMR spectra of enriched and isotopically normal ~-galactosidase are compared in Fig. 1. The e n z y m e in which '3C is in its natural abundance (1.1%) gives a very broad absorbance in the region approx. 10--30 ppm from TMS, the region o f alkyl carbons in t he protein. Enriched e n z y m e is characterized by a broad signal centered on 14.19 ppm, with width at half height A,,2 = 110 Hz, corresponding to the C c atoms of methionine. The average

148 TABLE 1 CHEMICAL SHIFT VALUES FROM TMS a

Ca C~ C~ Ce

Methionine

Methionine suifoxide

52.15

52.25 23.26 48.04 36.77

24.98 ; 28.67 14.13

a Measured in an aqueous 1 N HC1 solution at 20°C c o r r e l a t i o n time f o r the m e t h i o n y l m e t h y l groups calculated f r o m this value o f A~/2 is 2.1 • I 0 -9 s while r~ for the overall t u m b l i n g o f a rigid sphere the size o f ~-galactosidase is m u c h longer, 4 • 10 -7 s. In Fig. 2 spectra o f t h e samples a n a l y z e d in Fig. 1 are given after t h e y were l y o p h i l i z e d and dissolved in 70% a q u e o u s f o r m i c acid. E x c e p t for the r e s o n a n c e o f the C c a t o m in t h e e n r i c h e d sample the spectra are essentially identical, p r o v i n g t h e selectivity o f t h e e n r i c h m e n t p r o c e d u r e . The spectra in f o r m i c acid are c h a r a c t e r i z e d b y d i s t i n c t sharp signals instead o f a b r o a d signal as observed f o r t h e native e n z y m e , and b y a n a r r o w i n g C e signal A~/2 = 1 0 - - 2 0 Hz, f o r b o t h m e t h i o n i n e and m e t h i o n i n e sulfoxide. S p e c t r a o f ~3C-enriched ~-galactosidase are s h o w n in Fig. 3 at various stages o f o x i d a t i o n with c h l o r a m i n e T. T h e o b s e r v a t i o n o f a single p e a k increasing in i n t e n s i t y at 22.4 p p m d o w n f i e l d f r o m the ~3C-enriched m e t h i o nine m e t h y l p e a k c o n f i r m s t h a t t h e o n l y p r o d u c t o f this o x i d a t i o n o f m e t h i o n i n e is t h e sulfoxide. Details of t h e p r o c e d u r e used to d e t e r m i n e t h e ratio o f m e t h i o n i n e to m e t h i o n i n e sulfoxide residues are given in the legend

i i

•

i

a

•

,. ,..,..,_

b .... ~ ~':- I~/, •

40

~--~I

,II

• ~':If%

I k " .~

: , ~ I~I:L"

30

20 ppm

(from

10

0

TMS)

Fig. 1. a. t3C NMR spectrum at 67.9 MHz of 13C-enriched ~-galaetosidase at a concentration of 17.4 mg/ml, b. Spectrum of isotopically normal ~-galactosidase at a concentration of 22.6 mg/ml. Solutions were in sodium phosphate buffer (0.1 M, pH 7.0) containing 0.01 M magnesium chloride.

149

.

a

%,,

',, " ,../.4 '~..•.•• ---.:

" .../:-.

"..,•/.,.."

....

410

. . . .

3(3" " i

.:..

/.

20 ppm

(from

10

0

TM$)

Fig. 2. Spectra a and b are as in Fig. ] except that the enzymes were dissolved in 70% formic acid and enzyme concentration in spectrum a was reduced to approx. 10 mg/ml.

t o F i g . 3. T h e r a t i o in t h e s a m p l e t r e a t e d w i t h c h l o r a m i n e T w a s in g o o d agreement with the ratio determined by the chemical method [1]. This ratio can be converted to the actual number of residues of each type when the t o t a l m e t h i o n i n e in t h e s a m p l e is k n o w n . T h e r e s u l t s a r e s u m m a r i z e d in T a b l e 2. 1 8 o u t o f t h e 2 3 m e t h i o n i n e s o f f i - g a l a c t o s i d a s e a r e o x i d i z e d w i t h a molar excess of chloramine T of 2.15 and an even smaller molar excess of c h l o r a m i n e T ( 1 . 3 6 ) is s u f f i c i e n t t o o x i d i z e t h e f i r s t 11 r e s i d u e s . T h u s in /3-galactosidase a large proportion of the methionyl residues are readily accessible to the oxidizing agent. This, however, should not be interpreted t o m e a n t h a t all t h e o x i d i z e d m e t h i o n i n e s a r e e x p o s e d t o s o l v e n t s i n c e c h l o r a m i n e T c a n p e r h a p s r e a c h r e s i d u e s n o t d i r e c t l y a c c e s s i b l e t o s o l v e n t (cf.

[11). Since the broad natural abundance spectrum of the alkyl carbons shows n o s h a r p e n i n g t o i n d i v i d u a l p e a k s o n o x i d a t i o n o f t h e e n z y m e , it is u n l i k e l y

TABLE 2 METHIONINE SULFOXIDE OF fl-GALACTOSIDASE OXIDIZED WITH CHLORA-MINE T Chloramine T added (tool × 107)

Equivalent per subunit o f enzyme of reagent added

of methionine sulfoxide produced a

4.3 21.5 30.1 52.4

2.9 15.0 22.0 38.7

1 11 14 18

a The content of methionine sulfoxide was calculated from spectra a--d of Fig. 3 by the method described in the legend.

150

A

i,

g

j

a

, ."

~'~Y~

ii

,,k

i' i

L

!,

il fl /

d ..~.4W

i : ',,

~,

j v"

~

~¢~_~-~%

"V~

~'w-~.~

.

I!

II I

I

@ ..Mt~.j~, ],

-'-~

i

I

'

',~¢ .% ~.z

-' . . . . .

20

',~,

. . . .

~,.~'

"~. ,,V., ~' " , ~ ,

20 '

"

'

~'¢v,~-..-~,.....-~..-,-'..,,..,, .'. --.. u. -.

'

'

1'o

. . . . . .

0

ppm(from TMS) Fig. 3. S p e c t r a a--d are o f ~3C-enriched ~-galactosidase, 17.4 m g in 1.0 ml at pH 7.0 and v a r y i n g a m o u n t s o f a d d e d c h l o r a m i n e T: a, 0.43 ~ m o l ; b, 2.15 ~ m o I , c, 3.01 ~ m o l ; d, 5.24 ~ m o l . T h e c o n t e n t o f m e t h i o n i n e a n d of m e t h i o n i n e su]foxide were c a l c u l a t e d by i n t e g r a t i n g t h e areas of t h e spectra b e t w e e n 8 - - 2 8 p p m , (area A) a n d 3 0 - - 4 0 p p m (area B). Area A i n c l u d e s p e a k A ( 1 4 . 1 9 p p m ) w h i c h derives f r o m t h e C e a t o m o f m e t h i o n i n e as well as b a c k g r o u n d d u e to n a t u r a l a b u n d a n c e a b s o r p t i o n s . Area B consists of only the C e r e s o n a n c e s o f m e t h i o n i n e s u l f o x i d e ( 3 6 . 6 8 p p m ) , since n o b a c k g r o u n d a b s o r p t i o n is o b s e r v e d in this region (Fig. 1). T r a c i n g e in Figure 3 is o f t h e same s a m p l e w h o s e spect r u m is given in t r a c i n g d b u t a f t e r digestion w i t h p r o n a s e a n d s o l u b i l i z a t i o n w i t h 1 0 ~ f o r m i c acid. C o m p a r i s o n o f t h e ratios of i n t e g r a t e d areas A a n d B in s p e c t r u m d w i t h the areas u n d e r p e a k s A a n d B in s p e c t r u m e s h o w e d t h a t in native p r o t e i n 31% of t h e t o t a l i n t e g r a t e d a b s o r p t i o n in A derives f r o m t h e n a t u r a l a b u n d a n c e c a r b o n s . This area was s u b t r a c t e d f r o m t h e i n t e g r a t e d area A in e a c h native p r o t e i n s p e c t r u m t o give t h e true a b s o r p t i o n o f t h e e n r i c h e d 13ce r e s o n a n c e o f m e t h i o n i n e .

151

that treatment with chloramine T denatures the protein (see Fig. 3). Indeed we have shown that the catalytic parameters are changed but that the number of active sites is preserved in the oxidized enzyme [ 11]. SIMPLIFIED DESCRIPTION OF TIIE METHOD AND ITS APPLICATIONS 13C NMR of a protein in its native state can be used to determine the extent of oxidation of methionine of which the methyl carbon is enriched. Observation of 13CC resonances of methionine in a protein the size of ~-galactosidase (M r 460 000) suggests that this method will be applicable to most proteins. ACKNOWLEDGEMENTS

We thank Dr. M. Fridkin for his help with the synthesis of 13C-enriched methionine, Dr. R. Poupko and Mr. S. Rohald for their help with the NMR measurements and Mr. L. Esterman for his help with the sedimentation velocity measurements. REFERENCES 1 Shechter, Y., Burstein, Y. and Patchornik, A. (1975) Biochemistry 14, 4497--4503 2 Egan, W., Shindo, H. and Cohen, J.S. (1977) Annu. Rev. Biophys. 6, 383--417 3 Jones, W.C. Jr., Rothgebs, T.M. and Gurd, F.R.N. (1976) J. Biol. Chem. 251, 7452-7460 4 Eakin, R.T., Morgan, L.O. and Matwiyoff, N.A. (1975) Biochem. J. 152,529--535 5 Schejter, A., Lanir, A., Vig, I. and Cohen, J.S. (1978) J. Biol. Chem. 253, 3768-3770 6 Craven, G.R., Steers, E. Jr., and Anfinsen, C.B. (1965), J. Biol. Chem. 240, 2468-2477 7 Yariv, J. and Zipori, P. (1972) FEBS Left. 24,296--300 8 du Vigneaud, V., Dyer, H.M. and Harmon, J. (1933) J. Biol. Claem. 101, 7!9--726 9 Fowler, A.V., and Zabin, I. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1507--1510 10 Archer, R.A., Cooper, R.D.G. and Demarco, P.V. (1970) Chem. Commun. 1291-1293 11 Yariv, J., Kalb (Gilboa), A.J., Cohen, J.S., Jacobson, L. and Shechter, Y. (1978) in preparation