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Electrophoresis 1991, 12, 316-311

M. Chiari ptnl.

Marcella Chiari' Claudia Ettori' Pier Giorgio Righetti' Stefano Colonna' Nicoletta Gaggero' and Armando Negri3 'Departement of Biomedical Sciences and Technologies, University of Milano 'Department of Organic and Industrial Chemistry, University of Milano 31nstitute of Veterinary Physiology and Biochemistry, University of Milano

Oxidation of cysteine to cysteic acid in proteins by peroxyacids, as monitored by immobilized pH gradients It has often been debated whether the presence of persulfate in a polyacrylamide gel could lead to the oxidation of cysteine (Cys) in proteins to cysteic acid. In fact, direct incubation of bovine serum albumin (BSA) with peroxodisulfate and periodate barely alters the isoelectric point (pZ) and does not produce any cysteic acid. In contrast, caroate (peroxomonosulfate) and perphthalate strongly lower the p l ofBSA.In the formercase it as demonstrated that 4-Cys (ofa total of35) were converted into cysteic acid. Perphthalate was found to be, by far, the strongest oxidant: 15 (of 35) Cys residues were oxidized to cysteic acid and all methionine groups were destroyed.

When analyzing homogeneous preparations of recombinant prourokinase and urinary urokinase by isoelectric focusing (IEF) in immobilized pH gradients (TPG), in the pH 7-10 range, an extreme charge heterogeneity was detected. A great part of this polydispersity could be traced back to the existence of a multitude of protein molecules containing Cys residues at different oxidation levels (-SH and -S-S-) [ 11.The cause of these different redox states of Cys in urokinase was not apparent, although part of it might be attributed to spontaneous oxidation by atmospheric oxygen, owing to the relatively high plof native urokinase (ca. 9.59.8). It was subsequently discovered that, to a large extent, this oxidation power was inherent in the Immobiline matrix because, during the polymerization step, at appropriate pHvalues, the persulfate could oxidize, to different extents, all four alkaline Immobilines (the pK 6.2, 7.0, 8.5, and 9.3 species) used as buffers for focusing in basic pH regions [2]. It was suggested that, on exposure to ammonium persulfate, there would be addition of oxygen to the tertiary amino groups of deprotonated alkaline Immobilines, producing amine oxides (R,-N'O-). When focusing proteins in alkaline IPG ranges, free -SH groups would be oxidized to -S-S- bonds by the immobilized N-oxides, generating artifactual, higher p l bands. In a model system, in which free Cys was incubated anaerobically, at pH 9.0, with a crushed IPG gel, 100% oxidation to cystine was found in a 12-h period [3]. Such oxidation of free -SH groups in proteins (e.g. globin a-chains) could be demonstrated even in conventional IEF in the presence of soluble, amphoteric buffers, since these can also be oxidized by excess persulfate added during the polymerization step. So, even when the persulfate is discharged at the anode in a prefocusing step, potentially harmful oxidative power remains in the gel in the form of N-oxides of carrier ampholytes [4]. However, we were never able to find direct oxidation of Cys to cysteic acid, although this had been reported in the case of conventional IEF by Jacobs [51. Recently bovine serum albumin (BSA) was used as a chiral tool for the enantioselective oxidation of sulfides to the corresponding sulfoxides by in situ generated dioxiranes [6]. Similar results were obtained with potassium peroxomonosulfate (caroate) as oxidizing agent. Correspondence: Prof. P. G. Righetti, Department of Biomedical Sciences and Technologies, Via Celoria 2, Milano, 20133, Italy Abbreviations: BSA: bovine serum albumin; IPG, immobilized pH gradients; IEF, isoelectric focusing; PI, isoelectric point.

0VCH Verlagsgesellschaft mbH, D-6940 Weinhem, 1991

However, in both cases, it was found that the oxidantwas partially consumed in an ancillary reaction with the globular protein. This spurred us to investigate the possibility that, with this strong oxidizing agent, Cys in BSA could be oxidized to the irreversible state of cysteic acid. BSA (0.0375 mmol) was added at room temperature to 1.5 mmol of either caroate, or ammonium persulfate, or periodate, or perphthalate, or dioxirane at pH 7.4 and allowed to react for up to 24 h. In the case of caroate, BSA was also reacted in the presence of 0.75 mmol sulfide (paratolyl isopropyl sulfide). After gel filtration, reacted and control BSA was analyzed in an IPG, pH 4-8 range, prepared as in [7]. Additional aliquots were precipitated by 10% TCA, hydrolyzed in azeotropic HCI containing 1O h v/v phenol (gaseous phase) for 24 h at 110°C and then run through a Jasco automatic amino acid analyzer. The amino acid peaks were detected by postcolumn derivatization with o-phthaldialdehyde [8]. As shown in Fig. 1, hardly any change was detectable when BSA was incubated either with Na,S,O, or with periodate (although in the latter case some faint, more acidic bands were visible). On the contrary, upon incubation with caroate (KHSO,) the set of five BSA bands showed increased acidification, lowering the average plvalue by at least 1 pH unit. This suggested either suppression of positive charges, or introduction of new negative charges, or both. Interestingly, when BSA was incubated with caroate, but in presence of a sulfide (R-S-R), strong protection against band acidification was observed. The effect of perphthalate was even more pronounced: in this case, the average plwas lowered by ca. 2 pH units. Similar results are shown in Fig. 2, in which an additional oxidant was used, dioxirane (the smallest cyclic peroxide, produced by reacting caroate with acetone), which would appear to have somewhat more oxidizing power than caroate. Note that also in the case of dioxirane, incubation in presence of a sulfide strongly protects BSA (see Fig. 2, line 5). When subjecting BSA treated with the various peracids to amino acid analysis (Table l), it could be shown that, indeed, in the case of caroate, dioxirane, and perphthalate, Cys residues were progressively transformed into cysteic acid: ca. 12% with caroate (4 of 35 Cys residues), 18% with dioxirane (6 of 3 9 , and as much as 45% (15 of 35) in the case of perphthalate. In the last case, total destruction of Met was also found (see Table 1). No cysteic acid residues could be detected either in the control 0173-0835/91/0505-0376 $3.50+.25/0

Electrophuresis 1991, 12, 376-377

(Table 1) or in the case of persulfate- or periodate-treated BSA (not shown). There are two broad classes of organic peroxyacids: peroxycarboxylic acids [R(CO,H),] and peroxysulfonic acids (RSO ,OOH). Peroxycarboxylic acids are not usually utilized as free-radical initiators, unlike peroxysulfonic acids,

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Oxidation of Cys to cysteic acid

Table 1. Oxidation of Cys to cysteic acid in BSA Amino acid LY s His Arg Cys Aa’ Asxb’ Thr Ser Glx Pro GIY Ala CYS Val Met IIe Leu Tyr Phe Trp

Control

59 17 23 0 52 34 28 78 28 16 46 35 36 4 14 61 19 27 2

Caroate

55.0 15.7 24.5 4 58.3 32.7 24.8 76.1 ND~) 16.6 45.0 ND 28.4 2.0 12.4 63.1 18.7 27.5 ND

Dioxirane

Perphthalate

59 19.6 22.0 6 55 35.3 27.6 72.5 ND 17.9 46.4 ND 27.5 2.0 12.4 63.4 13 29.7 ND

53.6 19.6 21.7 14.9 55 36.9 29.3 77 ND 18.7 47 ND 27.5 0 12.8 62.6 11.2 29.5 ND

a) Cysteic acid b) Sum of Asx and some oxidation products of Met c) ND, not determined

F/gure I . Analysis o f BSA and its reaction products by IEF. Gel: 4%T, 4%cpolyacrylamide, containing an IPG, pH 4-8 interval, washed, dried, and reswollen in SM urea and 0.5% carrier ampholytes pH 4-8. Running conditions: 2000 V for 5 h at 10°C. The cathode is uppermost. Samples: 20 pg of BSA applied in 20 pL volume at the cathode. Ctrl, control, unreacted BSA, K H S 0 5 : BSA incubated with caroate alone orwith caroate plus a sulfide (paratolyl isopropyl sulfide; R-S-R); perphthalate: BSA incubated with perphthalate; NalO,: BSA incubated with periodate and Na2S208:BSA incubated with persulfate. Staining with Coomassie Brilliant Blue R-250.

such as ammonium persulfate (an almost universal initiator of polyacrylamide gel polymerization). Peroxyacids are the most powerful oxidizing agents of all organic peroxides. Typical oxidation reactions include epoxidation and hydroxylation of olefins and oxidation of sulfides to sulfoxides and sulfones; disulfides to thiosulfinates; and S-alkyl acetates to alkanesulfonic acids. Such oxidation reactions are generally accelerated by polar solvents [9]. However, it appears that only extreme oxidizing agents, such as caroate, dioxirane, and perphthalate, are able to oxidize Cys in proteins to cysteic acid (and also destroy Met, as in the case of perphthalate). No such reaction can be found with milder oxidizing compounds, such as persulfate and periodate. Supported in part bygrants,from Consigiio Nazionale delle Ricerche (CNR), Roma, Progetto Finalizzato Chimica Fine I I and by Agenzia Spaziale Italiana (ASI, PGR). Received January 22, 1991

References

Figure 2. Analysis of BSA and its reaction products by IEF. Gel: 4%T, 4%cpolyacrylamide, containing an IPG pH 4-8 interval, washed, dried, and reswollen In 8 M urea and 0.5% carrier ampholytes, pH 4-8. Running conditions: 2000 V for 5 h at 10’C. The cathode is uppermost. Samples: 20 1.18 of BSA applied in 20 pL volume at the cathode. Samples: (1) p l standards; (2) BSAcontrol, (3) caroate with a protective sulfide; (4) caroate alone; (5) dioxirane with a protective sulfide; (6) dioxirane alone; (7) perphthalate. Staining with Coomassie Brilliant Blue R-250.

[l] Righetti, P. G., Barzaghi, B., Sarubbi, E., Soffientini, A. and Cassani, G., J. Chromatogr. 1989,470, 337-350. [2] Righetti, P. G., Chiari, M., Casale, E. and Chiesa, C.,Appl. Theor. Electrophoresis 1989, I. 115-121. [3] Chiari, M., Chiesa, C., Righetti, P. G., Corti, M., Jain,T. and Shorr, R., J. Chromatogr. 1990,499, 699-711. [4] Cossu, G., Pirastru, M. G., Satta, M., Chiari, M., Chiesa, C. and Righetti, P. G., J. Chromatogr. 1989, 475, 283-292. [5] Jacobs, S . , Analyst 1973, 98, 25-33. [6] Colonna, S. and Gaggero, N., Tetrahedron Let. 1989, 30, 6233-6236. [7] Righetti, P. G., Immobilized p H Gradients: Theoty and Methodology, Elsevier, Amsterdam 1990, pp. 53-1 13. 181 Fujiwara, M . , Ishida, Y . ,Nimura, N., Toyama, A. and Kinoshita, T., Anal. Biochem. 1987, 166, 72-78. [9] Swern, D., Organic Peroxides, Wiley-Interscience, New York 1971.

Oxidation of cysteine to cysteic acid in proteins by peroxyacids, as monitored by immobilized pH gradients.

It has often been debated whether the presence of persulfate in a polyacrylamide gel could lead to the oxidation of cysteine (Cys) in proteins to cyst...
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