J. Biochem. 108, 432-440 (1990)

Reaction of Hen Egg-White Lysozyme with Tetranitromethane: A New Side Reaction, Oxidative Bond Cleavage at Glycine 104, and Sequential Nitration of Three Tyrosine Residues1 Hidenori Yamada, Taizo Yamashita, Hideki Domoto, and Taiji Imoto2 Faculty of Pharmaceutical Sciences, Kyushu University 62, Higashi-ku, Fukuoka, Fukuoka 812 Received for publication, April 20, 1990

We found that the reaction of hen egg-white lysozyme with an equimolar amount of tetranitromethane (TNM) at pH 8.0 and room temperature yielded derivatives in which the N-C bond of GlylO4 is oxidatively cleaved, and a mono-nitrotyrosine lysozyme in which Tyr23 is nitrated. This bond cleavage occurred more predominantly with a decrease in the nitration of Tyr23, when the reaction was carried out under more dilute conditions. A possible mechanism in which a phenoxyl radical of Tyr 23 (an intermediate of nitration) is involved was proposed for this oxidative bond cleavage. When lysozyme was reacted with a 10 times molar excess of TNM, in addition to a mono-nitrotyrosine lysozyme in which only Try23 is nitrated, a di-nitrotyrosine lysozyme in which Tyr20 and Tyr23 are both nitrated and a tri-nitrotyrosine lysozyme in which Tyr20, Tyr23, and Tyr63 are all nitrated were obtained. However, no other possible mono- or di-nitrotyrosine lysozymes could be isolated. Thus, it is concluded that the three tyrosine residues in lysozyme are essentially nitrated sequentially with TNM in the order of Tyr23, Tyr20, and Tyr53. Since the derivatives obtained here were all active, none of the three tyrosine residues or the residues around GlylO4 are considered to be very important for the lysozyme activity.

Although chemical modification is a useful means of revealing the role of a particular residue in the biological function of a protein, side reactions and protein denaturation are usually accompanied by chemical modification. Therefore, the results of chemical modification of a protein must be carefully interpreted. Tetranitromethane (TNM), a reagent described by Riordan et al. (1), has been widely used for the nitration of tyrosine residues to 3-nitrotyrosine residues in proteins (2). Hen egg-white lysozyme contains three tyrosine residues (Tyr20, Tyr23, and Tyr53) (3) and their nitration with TNM has been reported (4-6). Strosberg et al. (5) have demonstrated that the nitration of Tyr53 with TNM drastically decreases the activity of lysozyme against Micrococcus lysodeikticus (lytic activity), in contrast to the moderate decrease in lytic activity in the case of the nitration of Try20 or Tyr23. Since none of the three tyrosine residues is located in the active site cleft of lysozyme (7), the drastic decrease in activity due to the nitration of Tyr53 seems curious. Furthermore, in spite of the clear agreement in the literature that Tyr23 is the most reactive in the nitration of lysozyme with TNM at pH 8, there appears to be disagreement as to the second most 1 This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. * To whom correspondence should be addressed. Abbreviations: TNM, tetranitromethane; (NAG)j, y?(l-4)-linked trimer of Af-acetyl-D-glucosamine; TPCK-trypsin, L-l-(p-tosylamido)-2-phenylethyl chloromethyl ketone-treated trypsin; 23(NO), lysozyme, lysozyme derivative in which Tyr23 is nitrated; 20, 23-(NO,), lysozyme, lysozyme derivative in which Tyr20 and Tyr23 are both nitrated; 20,23,53-(NO,)3 lysozyme, lysozyme derivative in which Tyr20, Tyr23, and Tyr53 are all nitrated.

432

reactive tyrosine residue, Tyr20 (5) or Tyr53 (6). Therefore, we re-investigated precisely the reaction of lysozyme with TNM at pH 8 and room temperature (about 23'C). We found that these three tyrosine residues were essentially nitrated sequentially in the order of Tyr23, Tyr20, and Tyr53. Polymerization and fragmentation of lysozyme were detected in the precipitate formed in the reaction. Surprisingly, derivatives in which the N-C bond of GlylO4 was oxidatively cleaved were obtained as by-products. None of the derivatives isolated here exhibited drastically decreased activity. EXPERIMENTAL PROCEDURES Materials—Hen egg-white lysozyme [EC 3.2.1.17] was kindly donated by QP (Tokyo). Tetranitromethane (TNM) was obtained from Aldrich and used after washing with distilled water. M. lysodeikticus, a substrate of lysozyme, was obtained from Sigma. Glycol chitin, another substrate of lysozyme, was prepared as described elsewhere (8). A trimer of iV-acetyl-D-glucosamine, (NAG)3, was isolated from a partial acid hydrolysate of chitin according to the literature (9). Bio-Rex 70 (100-200 mesh) and Sephadex G-25 (coarse) were the products of Bio-Rad Lab. and Pharmacia, respectively. Analytical Methods—Amino acid analysis was performed on a Hitachi 835 amino acid analyzer after hydrolysis of a sample in 6 N HC1 under vacuum at 110'C for 20 h. The NH2-terminal sequences of peptides were determined with an Applied Biosystems Model 470A gas-phase protein sequencer. FAB mass spectra of peptides were measured with a JEOL JMS DX-300. Digestion of reduced and S-carboxymethylated lysozyme J. Biochem.

433

Oxidative Bond Cleavage during the Reaction of Lysozyme with TNM with L-l-(p-tosylamido)-2-phenylethyl chloromethyl ketone-treated trypsin (TPCK-trypsin, Sigma) and separation of the resultant tryptic peptides by reversed-phase high-performance liquid chromatography (HPLC) on a column of TSKgel ODS-120T (4.6x250 mm; Tosoh) were performed as described previously (10). Enzymatic activities of lysozyme derivatives against glycol chitin were determined in 0.1 M acetate buffer at pH 5.5 and 40°C (8). Activities against M. lysodeikticus were determined turbidometrically at 700 nm in 0.05 M potassium phosphate buffer at pH 7.0 and 25°C. RESULTS Reaction of Lysozyme with Tetranitromethane (TNM) —To a solution of 100 mg of lysozyme (7 /^mol) in 10 ml of 0.05 M Tris-HCl buffer (pH 8.0) was added 1.37 mg of TNM (7/^mol) dissolved in 1 ml of ethanol with stirring, and then the mixture was stirred at room temperature (23"C) for 2 h (run 1 in Table I). The reaction mixture was passed through a column of Sephadex G-25 (2.5x60 cm) with 0.05 M potassium phosphate buffer (pH 7.0) to remove trinitroformate formed. The protein fractions were combined, adjusted to pH 10.0 with a small amount of Na2B4O7 and 1 N NaOH, diluted with distilled water twice, and then applied to a column of Bio-Rex 70 (1.4 X 72 cm), a carboxylic acid cation-exchanger, which had been preequilibrated with 0.01 M borate buffer, pH 10.0. After loading, the column was eluted with a gradient formed from 1 liter of 0.02 M borate buffer (pH 10) to 1 liter of the same buffer containing 0.16 M NaCl. The chromatographic pattern obtained is shown in Fig. 1A. Besides the peak of unreacted native lysozyme (peak III), two yellow peaks (peaks I and II) and one colorless peak (peak IV) were obtained. The yellow color of peaks I and II indicated that the derivatives in these peaks (derivatives I and II) contain 3-nitrotyrosine residue(s). Assuming the molar extinction coefficient of lysozyme at 280 nm (e280) is 37,600 M~' •cm"1 (11) and that of a 3-nitrotyrosine residue at 381 nm (£38i, an isosbestic point) is 2,200 M"'-cm"' (2), the number of tyrosine residues nitrated was calculated to be 1.6 for derivative I and 0.9 for derivative II. Since a 3-nitrotyrosine residue absorbs light at 280 nm also (see below), the values obtained with this method should be underestimates. Thus, these results suggest that two tyrosine

residues in derivative I and one tyrosine residue in derivative II are nitrated. In order to increase the extent of nitration, 100 mg of lysozyme (7//mol) was reacted with a 10 times molar excess of TNM (13.7 mg, 70 //mol) for 30 h (run 7 in Table I). In this case, a considerable amount of lysozyme was precipitated during the reaction. After removal of the precipitate by filtration, the mixture was analyzed as described above. As shown in Fig. IB, a new peak (peak V) appeared together with peaks I and II. Unreacted native lysozyme (peak HI in Fig. 1A) and derivative IV (peak IV in Fig. 1A) were not obtained under these conditions. The derivative in peak V (derivative V) was also yellow and the extent of nitration was calculated to be 2.3 tyrosine residues per molecule from the ratio of the absorbance at 280 and 381 nm. As mentioned above, this value should be an underestimate and, therefore, all three tyrosine residues were considered to be nitrated in derivative V. All derivatives obtained here (derivatives I, II, IV, and V) were further purified by ion-exchange chromatography on Bio-Rex 70 at pH 7. All the derivatives except derivative IV only gave a single peak (data not shown), derivative IV being separated into two peaks (peaks IV-1 and IV-2 in Fig. 1C) in a ratio of about 7 : 1 . The derivatives thus purified (derivatives I, II, IV-1, IV-2, and V) were dialyzed against distilled water exhaustively and then lyophilized. Location of the Modified Residues in the Derivatives Formed during the Reaction of Lyosozyme with TNM— Table II summarizes the amino acid compositions of the derivatives isolated above. As expected, derivatives II, I, and V possess one, two and three 3-nitrotyrosine residues per molecule, with corresponding decreases in the numbers of intact tyrosine residues per molecule, respectively. The compositions of other amino acids of these derivatives were all the same as that of native lysozyme. These results clearly indicate that derivatives II, I, and V are mono-, di-, and tri-nitrotyrosine lysozymes, respectively. Surprisingly, derivatives IV-1 and IV-2, which were colorless, only had 11 glycines per molecule (there are 12 glycines in native lysozyme). As for other amino acids, the compositions of these derivatives were the same as that of native lysozyme. These results indicate that one out of the 12 glycine residues is destroyed but that tyrosine residues are all intact in these derivatives. In order to determine the locations of the nitrated

TABLE I. Reaction of lysozyme with TNM at pH 8.0 and room temperature." Run

TNM (//mol)

Vol. 108, No. 3, 1990

10 10

Additive'

Relation period (h)

Native (III)

23-NO, (II)

Yield" (mg) 20,23-(NO,)2 20,23,53-(NO,) (V) (I)

Nicked (IV)

Total

— 2 28.7 33.6 4.1 74.9 0 8.5 (NAG),, 2 0 22.1 40.5 6 . 3 71.5 2 . 5 10 mg 0 62.9 0 10 2 8 M urea 7 3 51.2 11.7 0 — 100 2 0 7 8.7 4 0 74.6 8.7 57.2 — 100 40 0 7 5 0 58.2 11.0 13.6 33.6 — 500 40 0 7 8.3 6 0 68.7 16.8 43.6 10 9.5 — 30 70 1.8 7 38.9 0 27.6 0 (NAG),, 10 12.0 40 70 8 57.5 0 43.9 1 . 5 0 10 mg 2 9 10 31.9 8 M urea 25.2 5.3 0 70 1.4 0 "To a solution of 100 mg of lysozyme (7 ^mol) dissolved in a given volume of 0.05 M Tris-HCl buffer (containing an additive) (pH 8.0) was added a given amount of TNM in 1 ml of ethanol with stirring, and then the mixture was stirred at room temperature for the indicated reaction period. "Recovery of each derivative and that of the total protein from the column of Bio-Rex 70 at pH 10.0. See Fig. 1, A and B. 1 2

7 7

Buffer volume (ml)

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H. Yamada et al.

tyrosine residues in derivatives II, I, and V, and those of the destroyed glycine residues in derivatives IV-1 and IV-2, these derivatives were reduced, S-carboxymethylated and then digested with TPCK-trypsin (10), and the resultant tryptic peptides were analyzed by reversed-phase HPLC.

aq at O

0.2

-

ELUTION VOLUME (liter) Fig. 1. Ion-exchange chromatography of lysozyme derivatives on Bio-Rex 70. A, reaction mixture of lysozyme with an equimolar amount of TNM (run 1 in Table I); B, reaction of lysozyme with a 10 times molar excess of TNM (run 7 in Table I); C, rechromatography of the protein in peak IV in A. In A and B, the column (1.4x72 cm) was eluted with a gradient formed from 1 liter of 0.02 M borate buffer and 1 liter of the same buffer containing 0.16 MNaCl.pH 10.0. In C, the column (1.4x70 cm) was eluted with a gradient formed from 1 liter of 0.05 M phosphate buffer and 1 liter of 0.3 M phosphate buffer, pH 7.0.

The elution patterns are shown in Fig. 2, B-F. For comparison, the pattern for native lysozyme is shown in Fig. 2A, where the assignment of each peak (12) is also shown. In the case of derivative II (Fig. 2B), native peptide T6 containing Tyr23 (Gly22-Lys33) disappeared and a new peak (peptide A) appeared between peptides T13 and T i l . T is used according to the nomenclature of Canfield (3) for the tryptic peptides of lysozyme. All other peptides were eluted at the same positions as the native peptides. The amino acid composition of peptide A was consistent with that of peptide T6 except that a 3-nitrotyrosine was substituted for a tyrosine (Table IS). Thus, derivative II is concluded to be nitrotyrosine 23 lysozyme (23-NO2 lysozyme). As for derivative I (Fig. 2C), in addition to the substitution of peptide A for native peptide T6, native peptide T5 containing Tyr20 (Hisl5-Arg21) was replaced by peptide B. The amino acid composition of peptide B was consistent with that of peptide T5 in which Tyr20 is nitrated (Table IS). Therefore, derivative I is concluded to be a di-nitrotyrosine lysozyme in which tyrosines 20 and 23 are nitrate [20,23-(NO2)2 lysozyme]. Similarly, derivative V is concluded to be a tri-nitrotyrosine lysozyme in which tyrosines 20, 23, and 53 are all nitrated [20, 23, 53-(NO2)3 lysozyme], as judged from the disappearance of native peptides T5, T6, and T8 (containing Tyr53, Asn46-Arg61), with the concomitant appearance of peptides A, B, and C (Fig. 3D), and from the amino acid composition of peptide C (Table IS). Derivatives IV-1 and IV-2 gave somewhat strange peptide elution patterns (Fig. 2, E and F). In both cases, native peptide T13 (Ile98-Argll2) disappeared and a new common peak (peptide F) appeared. Besides peptide F, peptides D and E from derivative IV-1, and peptide G from derivative IV-2 were eluted as new peaks. Peptide F, the common peptide, showed the amino acid composition of a peptide of MetlO5-Argll2 (Table IS). Peptide E from derivative IV-1 and peptide G from derivative IV-2 had the same amino acid composition, which was consistent with that of a peptide of Lys97-AsnlO3. As for peptide D, its

TABLE II.

Amino acid compositions of lysozyme and its derivatives.' Native lysozyme I rv-i n V IV-2 Theory Asp 21 20.3 20.2 20.2 20.2 20.4 20.3 Thr 6.7 6.7 6.7 6.9 6.9 7 6.7 Ser 9.1 9.0 8.7 10 9.1 9.1 9.5 Glu 5.2 5.4 5 5.3 5.3 5.3 5.4 Pro 2 2.1 2.0 2.0 1.9 1.9 2.0 Gly 12 12.0 10.8 (-1) 11.3 (-1) 12.0 12.0 12.0 Ala 12 12 12 12 12 12 12 Val 5.4 5.4 5.5 5.5 5.4 5.5 6 Met 2.2 2 2.0 1.9 1.9 1.9 1.8 He 5.6 5.6 6 5.5 5.4 5.5 5.6 Leu 8.0 8.1 8 8.0 8.0 8.1 8.0 1.3 (-2) 3.0 3 Tyr 3.0 2.0 ( - 1 ) 2.8 0.2 (-3) NO.-Tyr* 0 0 2.2 1.5 0 0 0.8 Phe 3.1 3.1 3.1 3 3.0 3.1 2.8 Lys 6.5 6.4 6.5 6 6.4 6.2 6.1 His 1.1 1 1.0 1.0 1.0 1.0 1.0 Arg 11 11.1 11.2 11.1 11.0 11.1 11.0 "All values are expressed as molar ratios as to a value of 12 for alanine. The values in parentheses indicate the estimated changes from native lysozyme. bSee Fig. 1. C3-Nitrotyrosine. The ninhydrin color yield of 3-nitrotyrosine was assumed to be the same as that of tyrosine. However, the values for 3-nitrotyrosine were somewhat smaller than expected. Thus, we supposed that the ninhydrin color yield of 3-nitrotyrosine would be about three-fourths that of tyrosine (see Table IS also). Amino acid

J. Biochem.

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Oxidative Bond Cleavage during the Reaction of Lysozyme with TNM Fig. 2. Reversed-phase HPLC of tryptic peptides obtained from reduced and S-carboxymethylated lysozyme derivatives on TSKgel ODS-120T. The column (4.6 X 250 mm) was eluted with a gradient formed from 40 ml of 1% acetonitrile and 40 ml of 40% acetonitrile, both containing 0.1% concentrated HC1, at theflowrate of 0.4 ml/ min. A, from native lysozyme; B, from derivative II (23-NO2 lysozyme); C, from derivative I [20,23-(NO,)i lysozyme]; D, from derivative V [20,23,53-(NO,)j lysozyme]; E, from derivative IV-1; F, from derivative IV-2.

100

TABLE III. Enzymatic activities of the derivatives obtained in the reaction of lysozyme with TNM. Lysozyme

Activity (%) against M. lysodeikticusb Glycol chitin" 100 100 84 92 65 93 60 91 94 76 88 73

Native 23-NO2 20,23-(NO2)2 20,23,53-(NOI), Derivative IV-1 Derivative IV-2 "In 0.1 M sodium acetate buffer at pH 5.5 and 40'C. "In 0.05 M potassium phosphate buffer at pH 7.0 and 25'C.

amino acid composition was consistent with a peptide of Ile98-AsnlO3. No peptides containing GlylO4 were detected at all. These results suggest that derivatives IV-1 and IV-2 are both nicked derivatives in which the main chain is cleaved at GlylO4 with the loss of this residue. The presence of nicks in these derivatives was also confirmed by the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (13) (data not shown). To clarify the difference between derivatives IV-1 and IV-2, peptide E from derivative IV-1 and peptide G from derivative IV-2 were further digested with TPCK-trypsin at 40'C for 4 h. Peptide E (Lys97-AsnlO3) was found to be digested to peptide D (Ile98-AsnlO3) with the release of Lys97. On the other hand, peptide G (Lys97-AsnlO3) was not digested at all. The lack of susceptibility of Lys97 in peptide G toward trypsin suggests that the e -amino group of Lys97 is also modified in derivative FV-2. Structures around the Nicked Sites in Derivatives IV-1 and IV-2—In order to determine the structures around the nicked sites in derivatives IV-1 and IV-2, the amino acid sequences and molecular weights of peptides D, E, F, and G were determined by gas-phase Edman degradation and Vol. 108, No. 3, 1990

200 RETENTION TIME (min)

100

200

FAB mass spectroscopy, respectively. The results and the elucidated structures of these peptides are summarized in Tables IIS and IIIS, respectively. As expected, the amino acid sequences of peptides D and E were consistent with those of the Ile98-AsnlO3 and Lys97-AsnlO3 peptides, respectively (Table IIS). Interestingly, the molecular weights of these peptides indicated that the C-terminal