Nitrosation of Tryptophan Residue(s) in Serum Albumin and Model Dipeptides

Nitrosation of bovine serum albumin with acidified NaNO 2 was compared to that of carboxymethyl-bovine serum albumin in which the thiol group is covalently blocked. Differential ultraviolet-visible (UV-Vis) spec- troscopy and a modified Saville assay indicated that a non-cysteine residue(s) in carboxymethyl-bovine serum albumin was nitrosated. The nitrosated carboxymethyl-bovine serum albumin exhibited similar vasorelaxation activity as that observed with nitrosated bovine serum albumin. Identification of the nitrosated non-cysteine residue(s) was studied using 16 model dipeptides, each of which contained a glycyl residue and a variable res- idue. Using photolysis-chemiluminescence analysis, modified Saville assay, differential UV-Vis spectroscopy, and bioassays, L -glycyl- L -tryptophan (Gly-Trp) was found to be the only dipeptide that underwent signifi- cant nitrosation under these conditions. Liquid chroma-tography-UV-Vis spectroscopy-mass spectrometry showed that the NO group was attached to the indole nitrogen of tryptophan. Nitrosated Gly-Trp exhibited dose-dependent vasorelaxation and platelet inhibiting activity with apparent EC 50 values of 1.1 (cid:54) 0.3 and 3.5

actions of nitric oxide (NO ⅐ ), a gaseous molecule that is generated by various mammalian cells and is involved in a variety of physiological and pathophysiological processes (3). Oxidation of NO ⅐ generates NO 2 and N 2 O 3 , which can, in turn, cause nitrosation. By contrast, S-nitrosation products, such as S-nitroso-N-acetylpenicillamine, S-nitrosoglutathione (GSNO), and S-nitroso-L-cysteine (Cys-NO), have been shown to induce similar biologic effects as that observed with NO ⅐ (4,5). Several studies have also demonstrated that NO ⅐ or S-nitroso compounds induce DNA mutations and changes in enzyme function by nitrosation (6 -10).
It has been assumed that the S-nitroso compounds induce NO ⅐ -like biological activity by releasing NO ⅐ through homolytic fission of the S-NO bond. Although such cleavage does occur under photolytic (11) or Cu 2ϩ (12) catalysis, a recent kinetic study demonstrated that the rates of formation of NO ⅐ from S-nitroso-N-acetylpenicillamine and Cys-NO are orders of magnitude smaller than the rates of transfer of -NO from these compounds to other thiols (13). Thus, -NO groups may reach their biological target via a transnitrosation pathway rather than by spontaneous release followed by nitrosation.
To explore the possible involvement of other amino acid residues in nitrosation reactions, we studied nitrosation of serum albumin and model dipeptides. S-Nitrosation of cysteine or cysteine-containing peptides (e.g. glutathione) has been previously studied in detail, and the resulting S-nitrosated derivatives produce vasorelaxation and platelet inhibition (4, 14 -16).
In this study, we examined whether or not other amino acid residue(s), in addition to cysteine, can be nitrosated and whether or not these nitrosated derivatives can manifest NO ⅐like activity. BSA was used as a model protein for this purpose and the nitrosation of carboxymethyl-BSA (CM-BSA), a modified derivative in which the thiol group is covalently blocked, was carried out. The identification and activity of the nitrosated non-cysteine residues were compared with those of 16 model dipeptides.
Nitrosation-NaNO 2 plus HCl were used as a nitrosating agent in this study. The combination of these two compounds will principally produce HNO 2 ; however, small amounts of N 2 O 3 and NOCl may also form in the reaction solution, and these species are stronger nitrosating agents than HNO 2 . For this reason, we use the denotation, NaNO 2 /HCl, throughout this study to describe the nitrosating species used.
With the exception of the time course experiments conducted with UV-Vis spectroscopic analysis, nitrosation was produced by first dissolving serum albumin (BSA and CM-BSA (200 mg/ml Ϸ3 mM)) or peptides (10 mM) in water; Gly-Trp was dissolved in 0.14 N HCl. Immediately before nitrosation, NaNO 2 (prepared in H 2 O at 50 mM) was diluted in 0.94 or 0.8 M HCl and mixed with an equal volume of protein (or peptide) solution. The final concentration of HCl in the mixture was 0.47 N. The reaction was carried out at room temperature for 30 -40 min and terminated by neutralizing the solution to pH 7.5 with 5 N NaOH in 0.5 M Tris buffer. The control samples (acid-treated) were prepared following the same procedure except that NaNO 2 was omitted from the reaction solution.
UV Visible Spectroscopy-Spectra were recorded at room temperature on a Cary 4E UV Visible spectrophotometer (Varian, Inc., Australia Pty., Ltd.). Protein or peptide was diluted in 0.5 N HCl and placed in both reference and sample cuvette. After correcting the baseline, NaNO 2 was added to the sample and an equivalent volume of H 2 O to the reference cuvette. Scanning was initiated immediately and 13 scans were performed at 5-min intervals. Computer-stored data (light transmittance) were converted to absorbance and plotted using a MicroCal Origin program (MicroCal Software, Inc., Northampton, MA).
Circular Dichroism-Circular dichroic (CD) spectra were recorded on an Aviv 62DS CD Spectropolarimeter (Aviv Associates, Lakewood, NJ) calibrated from 500 to 190 nm with D-10-camphorsulfonic acid (1 mg/ml in ethanol). Spectra were recorded at 25°C in 0.05-cm quartz cells from 250 to 185 nm with protein concentrations of 0.3 mg/ml. Multiple spectra (5-10) were recorded for duplicate samples prepared on different days. These spectra were averaged and corrected for baseline contribution from the buffer. Molar ellipticity values, [], were calculated according to the equation: [](degree cm 2 /decimal) ϭ ⌬ ϫ MRW/10 ϫ l ϫ c, where ⌬ is the displacement from the baseline value ϫ the full range in degree, MRW is the mean residue weight of the amino acids, l is the pathlength of the cuvette in cm, and c is the concentration of the protein in g/ml.
Data points were analyzed at 1-nm intervals between 250 and 190 nm by non-linear, constrained least-squares curve-fitting procedures to obtain estimates of each type of secondary structure. CD spectra were analyzed for ␣-helix, ␤-sheet, ␤-turn, and random coil using the LINEQ program of Cynthia Teeters as described by Mao and Wallace (17) utilizing the reference data sets of Greenfield and Fasman (18), Chang and colleagues (19), and Brahms and Brahms (20).
Thiol Content-Thiol content was measured according to a modified (21) Ellman assay (22). Briefly, protein or peptide was diluted in 0.1 M sodium phosphate containing 6 M guanidine HCl and 1 mM EDTA at pH 7.3. 5,5Ј-Dithiobis(2-nitrobenzoic acid) was added to a final concentration of 0.15 mM. The increase of absorbance at 412 nm was followed until maximal absorption was achieved (typically less than 5 min). The concentration of free thiols was calculated from the molar extinction coefficient of the nitrothiobenzoate ion in 6 M guanidine (⑀ 412 ϭ 13,700 Nitroso Content-Nitroso content was measured by two methods: Saville assay and photolysis-chemiluminescence analysis. In the Saville assay (23), the sample containing S-nitroso-derivatives was first mixed with 0.1% ammonium sulfamate in 0.4 N HCl (total volume ϭ 0.5 ml) for 1 min to remove NO 2 Ϫ or HNO 2 from the sample. A solution (0.5 ml) containing 3% sulfanilamide and 0.25% HgCl 2 in 0.4 N HCl was then added, followed by 0.5 ml of 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in 0.4 N HCl. The mixture was incubated at room temperature for 10 min and the absorbance was read at 540 nm. The nitroso content was calculated according to a standard curve constructed with 2.5-20 M NaNO 2 . (The ammonium sulfamate step was omitted in the standard curve measurements.) Photolysis chemiluminescence analysis was performed using a thermal energy analyzer (TEA model 543 analyzer, Thermedics Detection, Inc., Chelmsford, MA) equipped with a photolytic interface (Nitrolyte ® , Thermedics Detection Inc., Chelmsford, MA). The design of the instrument has been previously described (24 -26). Sample (100 l, 0.5-1 M peptide) was introduced into the system via an HPLC pump with H 2 O as the mobile phase (flow rate ϭ 1 ml/min). NO liberated from the sample by photolysis was carried by argon, separated from the liquid phase by condensation, passed into the thermal energy analyzer under vacuum, and mixed with ozone. Chemiluminescence resulting from the transition of NO 2 * to its ground state was detected and recorded, and the nitroso content calculated using a standard curve constructed using 100 -1,500 nM S-nitrosoglutathione as a standard synthesized by reacting glutathione with acidified nitrite, as described above.
Liquid Chromatography/Mass Spectrometry-Experiments were carried out using an atmospheric pressure ionization-electrospray liq-uid chromatograph/mass spectrometry (API-Electrospray LC/MS) system (Hewlett Packard Co., Palo Alto, CA), which consists of a HP-1090 liquid chromatograph, a HP 59987 API-Electrospray LC/MS interface, a HP 5989B MS engine, and a HP ChemStation data system with HP G1047A LC/MS software. A 2.1 ϫ 150-mm high surface area silica C 18 column (Vydac 201HS5215, Vydac Co., Hesperia, CA) was used for the HPLC separation. The samples were injected onto the column equilibrated with 10% acetonitrile, 90% H 2 O, and 0.1% trifluoroacetic acid (solvent A) at a flow rate of 0.3 ml/min. A linear gradient was started 5 min after injection to reach 90% acetonitrile, 10% H 2 O, and 0.1% trifluoroacetic acid (solvent B) in 40 min and then held at 100% solvent B for 5 min. The eluent was monitored at 275 and 335 nm by a diode array detector, which also recorded simultaneously the full UV-Vis spectra of the elution peaks. The eluent then entered a spray chamber with drying nitrogen gas at 245°C and passed through the capillary at an exit voltage of 72 or 150 V as indicated. Analyte ions were scanned over a range of 50 -800 m/z and the background was subtracted. Filtered raw data were presented without further refinement.
Vasorelaxation Assay-New Zealand White male rabbits (2.4 -3.6 kg) were sacrificed with pentobarbital (120 mg/kg) injected via a marginal ear vein. The aorta was excised and placed in ice-cold Krebs's buffer. Extraneous tissue and the vascular endothelium were carefully removed. The aorta was cut into 5-mm rings and suspended in an organ chamber (Radnoti Glass Co., Inc., Monrovia, CA) containing 10 or 20 ml of Krebs's buffer (37°C, pH 7.4) aerated with 95% O 2 , 5% CO 2 . The isometric tension was measured with a force transducer (Model FT-03, Grass Instrument Co., Quincy, MA). The vessel rings were prestretched with 6 gm tension and equilibrated for 90 min, after which the rings were contracted with phenylephrine (1 M). Relaxation was assayed following the addition of nitrosated peptides or proteins, prepared immediately before use.
Platelet Aggregation-Freshly obtained human platelets were kindly provided by the Naval Blood Research Laboratory (Boston, MA) and collected by mechanical apheresis, using the Haemonetics Mobile Collection System (Haemonetics, Inc., Braintree, MA) from donors who had not ingested any platelet inhibitor for at least 2 weeks. The blood was collected in acid-citrate-dextrose (ACS, NIH formula A) anticoagulant. The platelet-rich plasma was isolated by discontinuous-flow centrifugation with the Mobile Collection System. Platelets were counted with a Coulter Counter, model ZM (Coulter Electronics, Hialeah, FL), and diluted with platelet-poor plasma to a concentration of 1.7-2.0 ϫ 10 8 / ml. Aggregation was carried out on a PAP-4 aggregometer (Biodata, Hatboro, PA). Platelets were incubated with nitrosated or acid-treated (control) peptides/proteins for 15 min at 37°C. ADP (5-20 M, concentration required to achieve 80% aggregation response of control platelets) was added, and the platelets were stirred (1,000 rpm, 37°C) while aggregation was recorded. The extent of aggregation described in this report was recorded as the maximal extent of change in light transmittance.

RESULTS
Thiol Content and Effects of Nitrosation-We initially studied the nitrosation of three molecules, BSA, CM-BSA, and glutathione. Glutathione was used as a reference known to undergo stoichiometric nitrosation under these conditions with NaNO 2 /HCl (27).
The thiol (SH) content of these molecules was first examined. BSA had 0.41 Ϯ 0.11 mol of SH/mol of protein, while CM-BSA and glutathione had 0.02 Ϯ 0.004 and 0.98 Ϯ 0.08 mol/mol, respectively. The SH content in BSA was consistent with what has been published previously; i.e. the free cysteine residue in purified BSA is involved, in part, in mixed disulfide bond formation, and the most carefully prepared albumin contains at most 0.65-0.7 mol of -SH/mol of protein (28).
Owing to the possibility that acid could cause hydrolysis of disulfide bonds in proteins, the SH content of HCl-treated BSA was also determined. There was no significant change in SH content when the proteins were incubated with 0.5 N HCl for 40 min and then neutralized to pH 7.5, the conditions used for nitrosation.
BSA, CM-BSA, and glutathione were then nitrosated with equimolar NaNO 2 /HCl. Thiols of the nitrosated molecules were completely undetectable after the reaction.
UV Visible Spectroscopy- Figs. 1 and 2 show the time-de-pendent UV-Vis absorption spectra of a newly generated chromophore in glutathione, BSA, and CM-BSA during the course of reaction with NaNO 2 /HCl. In Fig. 1A, 13 spectra, collected over 1 h after mixing with equimolar NaNO 2 and glutathione in 0.5 N HCl, are plotted. The complete superimposition of these spectra indicated that the reaction of glutathione and NaNO 2 / HCl was rapid (completed within the mixing time of 20 -30 s), and the product was stable at acidic pH (0.5 N HCl). Increasing the NaNO 2 concentration 4-and 16-fold did not enhance the GSNO chromophore (Fig. 1, B and C), but at these molar excesses of NaNO 2 , the absorption peak of HNO 2 became apparent. HNO 2 absorption is illustrated by the tetrad of peaks between 300 -400 nM with max values of 347, 358, 371, and 386 nm (Fig. 1D); a small shoulder is also visible at 337-339 nm. The gradual decrease in chromophore intensity indicated that the initially formed HNO 2 underwent decomposition over the time course of these experiments (3HNO 2 The SH group of BSA's Cys 34 reacted promptly to produce a chromophore spectrum reminiscent of glutathione ( Fig. 2A); FIG. 1. UV-Vis absorption spectra of chromophore generation during the reaction of glutathione and NaNO 2 / HCl. After addition of NaNO 2 to the solution, spectra were recorded immediately and repeated every 5 min for a total of 13 cycles. Final concentration of glutathione was 0.3 mM in panels A-C, and the final concentration of NaNO 2 was 0.3, 1.2, 4.8, and 4.8 mM in panels A-D, respectively. Spectra were recorded against a blank containing glutathione in 0.5 N HCl (panels A-C). however, in contrast to glutathione, the absorbance continued to increase gradually over the hour of observation. When the maximal absorption of the first and the last scans was quantified (Table I), it became clear that the SH group in BSA (0.41 mol/mol) was nitrosated immediately after mixing with NaNO 2 (compare the molar absorption of BSA/NaNO 2 (1:1) with GSH/ NaNO 2 (1:1) at 0 min). The chromophore(s) generated over the subsequent hour of reaction and/or with higher concentration of NaNO 2 /HCl (Fig. 2, B and C) was likely derived from a different functional group, for which reason we next examined the nitrosation of CM-BSA.
The spectra of the newly generated chromophore in CM-BSA during nitrosation are shown in Fig. 2, D-F. The max of the absorption peak was observed at a shorter wavelength at the beginning of the reaction than at its completion, and at lower concentrations of NaNO 2 than at higher concentrations. As the reaction proceeded, or when higher NaNO 2 concentrations were used, the max shifted toward a longer wavelength, reaching a maximal absorbance wavelength at 334 -336 nm. Although the peaks observed were similar to those observed with BSA, the chromophore was likely derived from another reactive group owing to the fact that the SH group in CM-BSA had been covalently blocked. The more gradual increase in absorbance indicated the reaction was relatively slow, and the formation of an isosbestic point in the spectra as the absorption maximum shifted (Fig. 2D) suggested that a group migration had occurred. With neutralization of the pH at the end of the reactions, all three CM-BSA samples had absorption max of 335 nm, similar to that observed with BSA (data not shown).
The reaction of CM-BSA with NaNO 2 /HCl produced a bright yellow solution, while that with glutathione produced a reddish solution. Mixing BSA with equimolar NaNO 2 /HCl produced a light orange-red solution; however, at higher stoichiometries, the BSA solution became yellow and was similar to that of CM-BSA. The corresponding absorptions of these colors were not significant in the recorded spectra, likely due to their low extinction coefficients.
S-Nitroso Content-The similarity of the spectra of nitrosated BSA and CM-BSA at high NaNO 2 concentrations raised the question of whether or not the second reactive group was a thiol group generated by acid hydrolysis of disulfide. S-Nitroso content in these reaction products was thus analyzed, using the Saville assay. In the analysis listed in Table II, each sample was first depleted of free HNO 2 or NO 2 Ϫ by treatment with ammonium sulfamate, and then nitroso content measured with and without the addition of HgCl 2 . Since S-NO is stable at acidic pH, the -NO measured without HgCl 2 could not be derived from the S-NO group, but must be derived from other X-NO groups (X ϭ C, O, or N) that can spontaneously release -NO under acidic conditions. Table II, the S-nitroso content of BSA was 0.37 mol/mol. The S-nitroso content decreased with increasing NaNO 2 in the nitrosation reaction. A similar decrease was also observed with glutathione. However, in contrast to glutathione, the decrease in S-nitroso content was offset by an equivalent increase in non-S-NO content. Note that without neutralization, the S-nitroso content in nitrosated GSH was 0.97 (data not shown). CM-BSA did not contain detectable amounts of S-nitroso groups, but did have a measurable nitroso group at high NaNO 2 /HCl concentrations that could be detected in the absence of Hg 2ϩ . These data indicated that the nitrosated group in CM-BSA is a non-thiol group.

As shown in
CD Spectroscopy-To examine the secondary structure of the serum albumin species after their nitrosation and neutralization, far UV CD spectra of non-treated, HCl-treated and nitrosated BSA and CM-BSA were recorded and analyzed for specific elements of secondary structure. All spectra (data not shown) are characterized by negative minima at 222 and 208 nm and a positive maximum at ϳ190 nm, which are similar to data reported by others (29) and characteristic of a protein that possesses a predominantly ␣-helical conformation. All samples exhibit ϳ60% ␣-helix, ϳ40% ␤-sheet, ϳ4 -6% ␤-turn, and the remainder as random coil. Thus, neither nitrosation, HCl treatment, nor carboxymethylation alters the conformation or secondary structure of BSA.
Nitrosation of Model Peptides-In order to clarify which  functional group of BSA was nitrosated, studies on model peptides were carried out. Sixteen different dipeptides (Gly-Gly, Gly-Glu, Gly-Gln, Gly-Asp, Gly-Asn, Gly-Ser, Gly-Thr, Gly-Met, Gly-His, Gly-Pro, Pro-Gly, Arg-Gly, Gly-Lys, Gly-Phe, Gly-Tyr, and Gly-Trp) were examined. Among these peptides, the glycine residue was invariably included for the reasons of solubility and blocking of the ␣-amino group of the variable residue. The variable residues in the dipeptides included all the amino acids whose side chains contain a functional group in addition to hydrocarbon. Arg-Gly was used because of its commercial availability, and Pro-Gly was tested owing to the presence of a secondary amine group in the proline residue. Nitrosation of the peptides was carried out under the same conditions as those used for BSA, but the molar ratio between peptide and NaNO 2 was kept at 1:1. The degree of nitrosation of the dipeptides were estimated by three different methods: photolysis chemiluminescence analysis, Saville assay, and spectroscopic analysis. Among the 16 dipeptides tested, only nitrosated Gly-Trp released significant amounts (25%) of NO ⅐ after photolysis. Nitrosated Gly-Lys, Gly-Phe, and Gly-Tyr released 4, 5, and 6%, respectively. The remaining dipeptides released 1-3% NO ⅐ , similar to that of control, Gly-Gly, i.e. 3%. The residual signal is likely derived from the HNO 2 present in the reaction samples. Gly-Trp became yellow immediately after mixing with NaNO 2 /HCl. Nitrosation of Gly-Tyr also produced a chromophore with a yellow color, which appeared slowly after 20 min of reaction and darkened with neutralization of the pH. That no significant amount of NO ⅐ was released from the peptide by photolysis suggested that the color might be derived from a nitrophenol, rather than a nitrosophenol, structure.
The nitroso content of the peptides was also measured by the Saville assay without HgCl 2 . The reaction time with the Greiss reagents in this assay was extended to 30 min in order to detect slowly released nitroso groups. Nitrosated Gly-Trp was the only peptide that contained significant amounts (18%) of releasable nitroso groups. All of the other dipeptides contained 0% -NO. The lower content obtained in this assay from that measured by photolysis chemiluminescence analysis may be due to both incomplete release of -NO in the reaction and/or an effect of residual nitrite on the signal measured by the photolysis-chemiluminescence method.
UV visible spectroscopy was used to monitor the nitrosation process of the dipeptides. Three types of spectra (Fig. 3) were observed among the reactions of the 16 dipeptides. Nitrosation of Gly-Trp produced a chromophore with an absorption max of 335 nm at the onset of the reaction and a max of 316 nm at 60 min. Neutralization of the solution at the end of the reaction led to a reversion of max to 335 nm. The apparent molar absorption of the chromophore was 1684 M Ϫ1 cm Ϫ1 . The reaction of Gly-Tyr generated a wide absorption band at 258 -318 nm with an apparent molar absorption of 107 M Ϫ1 cm Ϫ1 (at 60 min) at 287 nm. The reactions of the other dipeptides showed similar spectra as that found for Gly-Phe. These spectra were equivalent to the HNO 2 spectra shown in Fig. 1D. From these spectra, it was concluded that the chromophore produced in the nitrosated CM-BSA was derived from a tryptophan residue in the protein. BSA is known to contain two tryptophan residues with one of them located in an aqueous solvent-exposed environment (30).
Reaction Products in Nitrosated Gly-Trp-In the peptide study described above and the biological activity study shown below, the reaction solutions of nitrosated peptide were used without separation of reaction products. To identify the reaction products in the solution, we analyzed the nitrosated Gly-Trp solution on a HPLC-coupled mass spectrometry system (API-electrospray LC/MS, see "Experimental Procedures").
As shown in Fig. 4A, the HPLC separation of nitrosated Gly-Trp revealed two major peaks with retention times of 8.7 (I) and 16.4 (III) min. Peak I exhibited identical UV-Vis spectrum and mass spectrum as that found in the control sample (acid-treated Gly-Trp, Fig. 4B). The mass ion at 262.6 m/z indicated that the molecule was a protonated form of Gly-Trp.
Peak III was identified as N-nitroso-Gly-Trp (N-NO-Gly-Trp) with the -NO group attached to the indole-nitrogen of tryptophan. This peak had UV-Vis absorption maxima at 267 and 335 nm and a shoulder at 273-274 nm, similar to that reported for N-acetyl-N 1 -nitrosotryptophan (267, 274, and 335 nm (31)). The two major ion species (261.3 and 187.4 m/z) in the mass spectrum were identified as the fragments of nitroso-Gly-Trp with cleavages at the N 1 -NO bond (261.3 m/z) and the N-C bond between tryptophan's ␣-carbon and ␣-amino residue (187.4 m/z), and the ion at 291.7 m/z was identified as the molecular ion. The low abundance of the molecular ion indicated that the homolytic N-NO bond fission had readily occurred during the electrospray process, which is consistent with a recent report that denitrosation occurs readily at high temperatures during electrospray-mass spectrometric analysis (32).
We also observed that N-NO-Gly-Trp had less structural stability than Gly-Trp. The fragmentation at the C-N bond (to generate the 187.4 m/z ion) occurred at a capillary exit voltage of 72 V in the N-NO-Gly-Trp sample but did not appear in Several other small peaks were also found in the HPLC elution profile (Fig. 4A). Peak II (14.8 min) was identified as N-nitro-Gly-Trp (N-NO 2 -Gly-Trp), which exhibited a major molecular ion peak at 307.4 m/z. The structure of two other minor peaks at retention times of 3.0 and 5.8 min were not identified. Both of the peaks had UV-Vis absorption maxima at 275-277 nm and no absorption at higher wavelengths. By mass spectral analysis, the 3-min peak showed a single ion species at 279.4 m/z and the 5.8-min peak showed two ion species at the 280.2 and 313.4 m/z. Vasorelaxation Activity-Vasorelaxation by nitrosated CM-BSA was examined and compared to that of S-nitrosated BSA. In this experiment, the proteins were reacted with equimolar NaNO 2 in 0.47 N HCl for 40 min and the pH neutralized to 7.5 immediately before the bioassay. As shown in Fig. 5, S-nitrosated BSA caused relaxation of aortic vessel rings by 13 Ϯ 14 and 71 Ϯ 22% at protein concentrations of 1.5 and 15 M, respectively. Nitrosated CM-BSA exerted less, but significant, relaxation: 5 Ϯ 7 and 63 Ϯ 21% at 1.5 and 15 M, respectively.
Dose-dependent Bioactivity of Nitrosated Gly-Trp-Vasorelaxation activity of all the NaNO 2 /HCl-treated dipeptides was examined. Nitrosated Gly-Trp was the only dipeptide that produced significant vasorelaxation, leading to 60.7, 81.5, and 90.5% relaxation at 1.5, 5, and 15 M, respectively. The other dipeptides produced relaxation in the range of 0 ϳ 3% at 1.5 M, 0 -8.5% at 5 M, and 7.2-18.8% at 15 M. The modest degree of nitrosation at the higher concentrations was likely a consequence of unreacted HNO 2 in the reaction mixture (see below).
To determine if the vessel relaxation caused by nitrosated Gly-Trp was exerted through a specific pharmacologic mechanism, the dose-dependence of relaxation was next examined. Nitrosated Gly-Trp was compared with three other compounds: acid-treated Gly-Trp, NaNO 2 , and S-nitrosoglutathione. As shown in Fig. 6, acid-treated Gly-Trp had no effect on vascular tone whereas nitrosated Gly-Trp exhibited a clear dose-dependent relaxation effect. The EC 50 for relaxation was calculated as 1.13 Ϯ 0.25 M, which was 2.5-fold less potent than that of GSNO (0.43 Ϯ 0.33 M). Because neither of the peptides were purified to homogeneity and the nitroso content of Gly-Trp and glutathione was approximately 22 and 89%, respectively, the actual potency of N-nitroso-Gly-Trp may be equal to or slightly greater than that of GSNO. NaNO 2 was significantly less potent than nitrosated Gly-Trp and GSNO; it produced 5, 17, and 41% relaxation at concentrations of 5, 15, and 50 M, respectively. The relaxations caused by all of the above compounds were reversible, indicating that no significant vascular toxicity occurred in the assay to account for the reduction in tone.
Inhibition of Platelet Aggregation-The effect of nitrosated Gly-Trp on platelet aggregation was also studied and compared to that of three other nitrosated molecules: glutathione, BSA, and CM-BSA. As shown in Fig. 7, all of these molecules produced dose-dependent inhibition of platelet aggregation. The IC 50 values for the nitrosated derivatives of glutathione, Gly-Trp, BSA, and CM-BSA were 0.7 Ϯ 0.3, 3.5 Ϯ 0.9, 1.3 Ϯ 0.3, and 17.3 Ϯ 7.9 M, respectively. Again, these values represent lower limits of the actual IC 50 values owing to the use of peptide or protein molarity rather than nitroso content. DISCUSSION In this study, a non-cysteine residue(s) in CM-BSA was found to be nitrosated by NaNO 2 /HCl. Using 16 model dipeptides, the non-cysteine residue was characterized and the structural properties indicated that the residue was tryptophan. Nitrosated CM-BSA and Gly-Trp exhibited NO ⅐ -like vasorelaxation and antiplatelet activity, similar to that of Sdescribed under "Experimental Procedures." The eluent was analyzed subsequently by a diode array detector and an API-electrospray LC/MS system. Recorded HPLC chromatograms are shown in the top row, and UV-Vis spectra and mass spectra of the major HPLC elution peaks are shown in middle and bottom rows, respectively. . The reagents were prepared by incubation of 5 mM each of NaNO 2 and peptides in 0.5 N HCl at room temperature for 40 min, followed by neutralizing to pH 7.5. The NaNO 2 control did not contain peptide and acid-treated Gly-Trp control did not contain NaNO 2 . Relaxation is expressed as percent decrease in tension. All values given are mean Ϯ S.D. with n ϭ 8 animals in each group. The difference between each group was significant (p Ͻ 0.05) by two-way RMANOVA.
Although tryptophan was the only non-cysteine residue that showed significant nitrosation in our experiments, other amino acid residues, such as arginine, lysine, asparagine, glutamine, and tyrosine, may have undergone initial nitrosation to a small extent. However, nitrosation of the primary amine group in arginine, lysine, asparagine, and glutamine is known to be followed by diazotization and denitrogenation; and nitrosation of tyrosine is followed by the irreversible oxidation of -NO to -NO 2 (33,34). Thus, the initially formed nitroso groups in these residues were likely eliminated by the secondary reactions and they were consequently not detectable. To our surprise, we could not detect the nitrosation of proline, whose product is known to be stable. Since our detection methods included a physical cleavage of the NO group using UV energy sufficient to cleave the N-NO bond, the inability to detect nitrosoproline most likely implies that it did not form under our experimental conditions. Previous kinetic data suggested that the formation of nitrosoproline is slower than that of N-acetyl-N 1 -nitrosotryptophan (35,36).
Tryptophan is known to undergo nitrosation and denitrosation. The equilibrium constant for the formation of N-acetyl-N 1 -nitrosotryptophan was found to be 850 M Ϫ1 (36). Mechanistic studies indicated that intramolecular migration of the -NO group from C-3 to N-1 in the tryptophan indole ring occurs during the nitrosation of N-acetyltryptophan, and this event is the rate-limiting step of the reaction (36,37). Our observation of isosbestic point formation in the nitrosation spectra of Gly-Trp and CM-BSA may reflect such an intramolecular migration of -NO.
Compared to the nitrosation of cysteine, nitrosation of tryptophan is significantly slower and the product is less stable. As shown in our spectroscopic characterization, nitrosation of glutathione with NaNO 2 /HCl was completed within 20 s (mixing time), but nitrosation of tryptophan reached a maximum (18 -25%) after 5 min. Using slowly delivered NO ⅐ gas as nitrosating agent (NO ⅐ /argon mixture diffused through Silastic tubing and into a solution at pH 7.4 under aerobic condition), our preliminary data showed that the formation of N-nitroso-Gly-Trp is approximately 10-fold slower than the formation of GSNO, and the latter is 2-3-fold slower than the formation of nitrite. Thus, in a spontaneous nitrosation reaction, tryptophan cannot com-pete favorably with cysteine. In addition, tryptophan nitrosation has other characteristics different from cysteine nitrosation. For example, nitrosation of tryptophan is a completely reversible reaction, but nitrosation of cysteine is rarely reversible because denitrosation of S-NO-cysteine usually produces an oxidized form of cysteine (cystine). If both cysteine and tryptophan residues are involved in a repeated transnitrosation process, the former will require a reduction system to recycle the cystine, but the latter will not.
The biological effects of N-nitrosotryptophan have been reported in terms of its mutagenicity. Using synthetic N-acetyl-N 1 -nitrosotryptophan, Venitt and colleagues (38) showed that the compound causes mutation in a series of E. coli WP2 strains (trp Ϫ to trp ϩ ) and several Salmonella typhimurium strains (his Ϫ to his ϩ ). Since a similar effect has been observed with NO ⅐ gas (39,40), these data observations demonstrate that N-nitrosotryptophan, in addition to mediating vasorelaxation and antiplatelet aggregation, can produce other NO ⅐ -like biological effects.
How extracellular N-nitrosotryptophan causes intracellular biological effects has not been investigated. It is obvious that this molecule cannot freely diffuse through cellular membranes and does not spontaneously release NO ⅐ radical via homolytic N-NO bond fission. Therefore, to mediate its NO ⅐ -like effects, the -NO group on the N-nitrosotryptophan (either N-NO-Gly-Trp or nitrosated CM-BSA) must be transferred to guanylyl cyclase (for vasorelaxation) or DNA (for mutation) via intermediate -NO carrier(s), and one of these must be located on or associated with the plasma membrane. Interestingly, such a carrier does not seem to be strictly selective regarding the type of nitroso group because the potency of N-NO-GlyTrp was similar to that of S-nitrosoglutathione. There is little current knowledge about the biochemical mechanism(s) of transnitrosation, although such a mechanism appears to be relevant for the biologic actions of nitroso compounds.