A New Method for Determining Sulfoxides in Peptide Molecules Using X-Ray Photoelectron

Spectroscopy*

DEREK JONES and GIUSEPPE DISTEFANO, Laboratorio dei Composti del Carbonio Contenenti Eteroatomi e loro Applicazioni,

C.N.R., 40064 Ozzano Emilia, Bologna, Italy; and CLAUD10 TONIOLO and GIAN MARIA BONORA, Centro di S tudi sui

Biopolimeri, C.N.R., Zstituto di Chimica Organica, Uniuersitci di Padoua, Padua, I taly

Synopsis

The problem of the quantitative determination of sulfoxide groups in peptide molecules has been re-examined. The approaches currently available for the estimation of 6-sulfoxide amino acids are limited in number and characterized by serious shortcomings; in addition, the choice of methods for the estimation of y-sulfoxide amino acids is even more restricted. A new, rapid, and nondestructive direct method for determining quantitatively all types of sulfoxides in peptide molecules by using x-ray photoelectron spectroscopy is described.

INTRODUCTION

X-ray photoelectron spectroscopy (XPS) is a technique for determining the kinetic energies of electrons photoejected by x-rays2 The ionization (or binding) energies of inner-shell electrons are obtained from the dif- ference between the known energy of the exciting radiation and the mea- sured kinetic energy of the emitted photoelectrons. The resulting spectra directly reflect the orbital structure of the elements present in the sample. With the exception of hydrogen, all elements can be determined. From the chemical shift of the photoelectron lines with respect to reference compounds, conclusions can be drawn concerning the charge, the oxidation state, and the chemical environment of an atom.

This technique has been extensively used over the last few years to gain information on metalloproteins, where XPS is particularly suitable for those metal ions which cannot be investigated using magnetic susceptibility, electron spin resonance, or Mossbauer spectroscopy.

The NIHg and SIHg ratios have been directly measured with 5-10% accuracy in mercuripapain (approximate molecular weight: 42,000), which contains one atom of m e r ~ u r y . ~ In cases where the direct measurement of a functional group in the protein is difficult, specific modification with groups containing atoms which give an intense XPS response can be used to estimate the functional group content of a protein.3

* This is part 47 of the series Linear Oligopeptides. For part 46, see Ref. 1.

Biopolymers, Vol. 17,2703-2713 (1978) 0 1978 John Wiley & Sons, Inc. 0006-3525/78/0017-2703$01.00

2704 JONES ET AL.

XPS has been shown to be a valuable tool in determining the integrity of the Fe-S bond in cytochrome c , ~ while a strong polarization of sulfur bonded to mercury has been proposed from a comparison of Cd- and Zn- thionein and Hg-thionein.5

The valency of iron in ferredoxins has been e~tablishedF9~ while the presence of satellites in the Cu2p lines of several complexes of biological significance? and the detection of an exchange interaction of Fe& electrons with unpaired 3d electrons in ferredoxinsg have clearly demonstrated that the metal ions are individually in magnetic states, even in compounds wi- thout magnetic moments, because of antiferromagnetic coupling between pairs of metal ions.

Remembering that XPS is a surface technique, the observed intensity ratio of the Znzp3/2 and CuzP3/2 signals in bovine erythrocuprein, being much lower than the expected ratio, lead to the conclusion that the cupric ions are most probably located on the surface portion of the protein, whereas Zn2+ appears to be bound in the inner sphere of the protein.1°

More than one type of sulfur has been observed in several cases: the modification of bovine serum albumin with ethylvinyl sulfone has been observed and measured1+ two or three sulfur species have been shown to exist in metallothioneins (S2, from RS-, R-SS-R, and RS03-)12; the thiosulfonate structure of cystine-S-dioxide has been proved13; and the disulfide and sulfonate oxidation states of sulfur have been detected in insulin selectively oxidized with iodate.2

In this paper we describe a new, rapid, and nondestructive direct method for determining quantitatively all types of sulfoxide groups in peptide molecules (e.g., those occurring in the y- and &positions of amino acid side chains) in the presence of thioether groups using XPS. Factors affecting the application of this technique to quantitative analysis have been dis- c~ssed.~.3J4

MATERIALS AND METHODS

Materials

L-Methionine, S-methyl-L-cysteine [Cys(Me)], and N-chlormuccinimide (NCS) were purchased from Fluka A. G., Buchs. p-Toluenesulfonic acid monohydrate was obtained from Pierce Chem. Co., Rockford, 111. Boc- (~-Met)2-s-OMe and Boc-(L-Cys(Me)-)24-OMe have been synthesized as described in Refs. 15 and 16, respectively. The sulfoxide derivatives of L-methionine and S-methyl-L-cysteine have been prepared by treatment of the thioether amino acids with equimolecular solutions of N-chlo- rosuccinimide in 50% acetic acid at room temperature for 15 min.17 The R f values (ascending TLC; Si02, Merck) in n-butanollacetic acid/water (602020) of the two sulfoxide amino acids were 0.15-0.20, in contrast with the values of 0.40-0.50 for the two thioether amino acids. The optical rotation values observed for methionine sulfoxide, L-Met(O), and S-methyl cysteine sulfoxide, L-Cys(Me)(O), indicated that both sulfoxides were

X-RAY METHOD FOR PEPTIDE SULFOXIDES 2705

obtained as mixtures of the two corresponding epimers at the chiral sulfur atom. 18719

Methods

The amino acid analyses were performed on a C. Erba amino acid ana- lyzer, model 3A27, in the standard conditions of analysis for neutral and acidic amino acids.

The XPS spectra were obtained with an AEI ES 200B photoelectron spectrometer operated at a pressure of about torr. The energy of the exciting x-rays was 1486.6 eV (A1 Kal,2 radiation) with a power of 150 W. The samples were introduced on clean metal surfaces as thin films obtained by evaporation of small amounts of ethanol/water solutions of the amino acid or peptide, minimizing the quantity of water used as the latter tended to broaden the bands observed. When introducing the solids directly, the observed signal from the sulfoxide was always about 25% of its weighed value relative to that of the sulfide. This was thought to be due to the sulfoxide particles sticking to each other and being covered by particles of the sulfide, thus reducing the observed sulfoxide/sulfide intensity ratio. At least four measurements were carried out on each sample and the relative concentrations of sulfoxide/sulfide present were estimated by hand de- convolution of the two corresponding bands in the SBP region of the XPS spectrum, taking as reference the shape of the S2p bands in pure L-Cys(Me) and L-Cys(Me)( 0), respectively. The results were expressed as relative percentages of the two types of sulfur present. These values were fairly reproducible (mean deviation: 1.5% units). None of the samples showed noticeable signs of decomposition during the measurements under the conditions used. No impurities, except 01, and CIS, were observed. Sulfone derivatives were also absent. The minimum quantity of sample required for a measurement was of the order of 10 pg.

RESULTS AND DISCUSSION

In recent years the sulfoxide amino acids have attracted increasing at- tention among peptide and protein chemists. The oxidation state of the sulfur influences properties such as the configuration, solubility, and pK values, and changes in these properties may in turn alter the chromato- graphic and ionophoretic behaviors of the polypeptides in which they occur. Amino acid residues containing sulfur in thioether linkage in y-position (e.g., S-alkyl-cysteines) and in &position (e.g., methionine) may be readily oxidized to the corresponding sulfoxides under mild conditions. Attempts to isolate ACTH, and a- and P-MSH, frequently resulted in formation of inactive Met(0) derivatives.20 In proteins, photooxidation and reaction of aromatic amino acids with electrophiles often gives rise also to oxidation of thioether sidechains.21>22 Sulfoxide amino acids have been found in byproducts in the course of peptide synthesis.23 To avoid alkylation of thioether amino acids in peptide synthesis they are sometimes incorporated

2706 JONES ET AL.

MINUTES MINUTES

Fig. 1. Amino acid chromatograms. (A) 0.15 pmol L-Met, L-Met(O), and L-G~u (as internal reference). (B) Mixture A following treatment with 3N p-toluene-sulfonic acid (llO°C, 22 hr). (C) 0.15 pmol L-Cys(Me), L-Cys(Me)(O), and L-G~u (as internal reference). (D) Mixture C following treatment with 3N p-toluene-sulfonic acid (llO°C, 22 hr).

into the peptide chain as sulfoxide derivative^.^^ The biological and spectroscopic properties of synthetic analogs or derivatives of peptides and proteins containing sulfoxide amino acids have been compared to those of natural comp0unds.~5,~6 Sulfoxide amino acids have been used as oxidizing

and they also occur in natural p r o d ~ c t s . ~ ~ ~ ~ ~ The use of di- methylsulfoxide (in the presence of catalytic amounts of hydrogen halides) as solvent in nmr measurements can result in the oxidation of thioether amino a ~ i d s . ~ ~ ? ~ ~ Penicillin and cephalosporin sulfoxides are important intermediates in the synthesis of cephalo~porins.~~

The amounts of 6-sulfoxide amino acids in peptides and proteins can be determined either directly by chromatography after alkaline hydrolysis or by indirect method^.'^.^^ The latter depend on the fact that sulfoxide amino acids, in contrast to thioether amino acids, are resistant to reaction with electrophiles (e.g., iodoacetic acid, cyanogen bromide, etc.). Acid hydrolysis (with hydrogen halides) is useless since 6-sulfoxide amino acids are converted to the corresponding thioether derivative^.^^,^^ Although analysis after alkaline hydrolysis is the fastest and more direct approach, it has at times resulted in low recoveries of 6-sulfoxide amino acids.22 In addition, a number of amino acids are obtained in low yield due to de- struction by the alkali.Z2 Complications often also arise in the use of in- direct methods, since their successful application is dependent on the complete reaction of the unoxidized 6-thioether amino a ~ i d s . ~ ~ , ~ ~ The choice of methods for the estimation of y-sulfoxide amino acids is even more restricted35-37 (for instance cysteine and its derivatives are labile at alkaline PH).

X-RAY METHOD FOR PEPTIDE SULFOXIDES 2707

t s

I

-5- a

- 5 0 -

K I N E T I C E N E R G Y (eV) - - Fig. 2. XPS spectra of SzP electrons of L-Cys(Me) (A), mixtures of L-Cys(Me) and L-

Cys(Me)(O) containing 17% (B), 46% (C), 79% (D), and 91% (E) of L-Cys(Me)(O); and L- Cys(Me)(O) (F).

Recently, it has been reported that Met(0) can be quantitatively de- termined in peptides and proteins by acid hydrolysis22 using 3N p-tolu- enesulfonic acid (llOOC, 22 hr).38 We have re-examined the behavior of Met(0) and investigated that of Cys(Me)(O) in 3N p-toluenesulfonic acid (llO"C, 22 hr) in the presence of Met and Cys(Me), respectively. The pertinent amino acid chromatograms are shown in Fig. 1. It is evident that this method can be employed, although with caution, for the quantitative determination of Met(O), whereas it is useless for the estimation of Cys(Me)(O). It has to be noted that the recovery of the thioether amino acids under these experimental conditions is also reduced. Glutamic acid was used in this investigation as an internal reference. The elution times of thioether and sulfoxide amino acids under the standard conditions of

2708 JONES ET AL.

0 1 I I I I I I I I

0 20 40 60 80 100 % SULFOXIOE ( c a l c u l a t e d )

Fig. 3. Plot of observed sulfoxide percentages (from XPS) vs calculated sulfoxide per- centages (by weight) of mixtures of L-Cys(Me)(O) and L-Cys(Me).

analysis of neutral and acidic amino acids have previously been reported.39 The peaks corresponding to sulfoxide amino acids are always split into two components because of the presence of epimers at the sulfur chiral atoms (see Materials and Methods).

From the discussion and the results reported above, it is clear that none of the methods currently employed for the quantitative estimation of sulfoxide amino acids can be considered entirely satisfactory. In addition, all of them are destructive in character. Therefore, we decided to apply XPS to this problem.2JJ4

Figure 2 shows the XPS spectra of sulfur 2p electrons of L-Cys(Me), L-Cys(Me)(O), and mixtures containing increasing amounts of the latter amino acid residue. The peak-to-peak separation in the sulfur 2p region of the spectra of these mixtures is 2.6 f 0.1 eV; meanwhile, the N1,/S2, separation increases by 3.0 f 0.1 eV on going from L-Cys(Me)(O) to L- Cys(Me). This small but reproducible discrepancy could indicate a weak intramolecular interaction between sulfoxide and a-ammonium groups. The corresponding increase in the N,,/Sz, separation in the pair L-Met, L-Met(0) is only 2.5 f 0.1 eV (see below), in accordance with the lack of such neighboring-group effects,4O and probably explained by the increased number of methylene units between the two groups. From our spectra we were not able to distinguish between diastereomeric sulfoxides. The electron binding energies of Cys(Me) and Met have previously been re- p0rted.~l-45

The general utility of XPS as a quantitative tool in the determination

X-RAY METHOD FOR PEPTIDE SULFOXIDES 2709

- K I N E T I C ENERGY (ev) -. Fig. 4. Amino acid chromatogram (A) and Sz,, region of XPS spectrum (B) of a mixture

of L-Cys(Me) and L-Cys(Me)(O) prepared by partial oxidation of L-Cys(Me) with N-chlo- rosuccinimide.

of sulfoxide groups in the presence of thioether groups is demonstrated by the calibration curve illustrated in Fig. 3. However, the numerical values of the sulfoxide percentages found were usually slightly lower (04% units) than those expected. Considering the possibility that the chemical form of the sulfur atom could affect the relative intensity of the SzP peak in the two compounds,14 the Ssp/Nls intensity ratio for both was checked and found to be identical (1.23). The origin of the slightly low sulfoxide in- tensity could be found in the hygroscopic nature of L-Cys(Me)(O), which would produce such a weighing error.

To demonstrate the utility of XPS for our quantitative analytical pur- pose, the calibration curve shown in Fig. 3 was used to determine the sulf- oxide percentage in a sample prepared by reacting L-Cys(Me) with NCS (-0.6 equivalents) in 50% acetic acid a t room temperature for 15 min.17 Both amino acid analysis and XPS (Fig. 4) revealed that the sample was made up of 63% Cys(Me)(O) and 37% Cys(Me).

In the Padua laboratory, two of us (C.T. and G.M.B.) have recently synthesized the monodisperse N- and C-protected homooligopeptide series, from dimer through to heptamer, derived from ~-Met'5 and L-Cys(Me)'G

2710

t P

I

JONES ET AL.

- S- f C 0 ) NH-

238.1

I , I

I NHa-

- K I N E T I C E N E R G Y (‘V) - Fig. 5. XPS spectra of N1, and S2p electrons of Boc-(L-Met)z-OMe (A), L-Met (B), and

L-Met(0) (C).

X-RAY METHOD FOR PEPTIDE SULFOXIDES 2711

for a stereochemical investigation. Since the chemical purity of oligo- peptides is of paramount importance for the onset of stable secondary structures, we have examined some representative examples of the two series (dimers through to pentamers) by XPS to check if any oxidation at the thioether sulfur atoms had taken place. The XPS spectrum of Boc- (~-Met)z-OMe is shown in Fig. 5(a). All homooligopeptides examined have been found completely free of sulfoxide impurities. Also, the XPS spectra do not seem to be sensitive to changes in the secondary structure of our homooligopeptides (from a random-coiled conformation to a P-confor- m a t i ~ n ) . ~ ~ In fact, the energy differences between the SzP line and those of the other atoms (Cl,, Ols, and N1,) are constant within experimental error in the Cys(Me) series. Also, the form of the N1, and S2p bands is constant, whereas the 01, band shows a smaller half-width in the pentamer, as do the C1, lines in the trimer and pentamer, which is accompanied in both cases by a reduction in the high-binding-energy shoulder from the oxidized carbon atoms (CO and COO). Because of the presence of background 01, and C1, signals, these effects are difficult to assess. Figure 5 also illustrates the N I , / S ~ ~ peak-to-peak separation of L-Met(O), Boc-(~-Met)z-OMe, and L-Met; from that of L-Met(O) (236.9 eV), we observed an increase in the separation in the expected d i r e c t i ~ n ~ , ~ ~ of 1.2 and 2.5 eV on going to Boc- (~-Met)z-OMe and L-Met, respectively.

To summarize, we have demonstrated that all types of sulfoxide groups (e.g., those occurring in the y- and &positions of amino acid side chains) can be quantitatively determined in peptide molecules in the presence of thioether groups using XPS. Although XPS analyzes only the surface of solids (about the uppermost 100 A), it is our contention that this surface analysis is representative of the bulk sample in this case. However, it is pertinent to recall that the application of XPS to quantitative analysis must be carried out with caution, since the use of different compounds in these studies might result in vastly different sensitivities. In any case, the spectrometer must be rigorously calibrated before use.

This direct approach, compared to amino acid analysis and indirect analytical method^,'^,^^ is (1) much more rapid, since the time needed for the XPS measurements, including sample preparation, is 1-1.5 hr; and (2) nondestructive, since radiation damage to the sample can be avoided by short run times.47 In addition, the reproducibility (mean deviation: 1.5% units), accuracy (-5% units), and the quantity of sample required for a measurement (((1 mg) all favor the proposed analytical approach compared to indirect method^.^^,^^

The authors wish to thank Drs. A. Fontana and E. Bordignon, Centro di Studi sui Biopo- limeri, CNR, Istituto di Chimica Organica, University of Padua, for stimulating discussions and for making available results from their laboratories prior to publication.

2712 JONES ET AL.

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Received August 8,1977 Accepted February 6,1978