Nitration of Tyrosine Residues in the Pancreatic Trypsin Inhibitor with Tetranitromethane

Abstract

Nitration of pancreatic trypsin inhibitor with tetranitromethane affords a mono-and a disubstituted derivative which was shown by sequential analysis to contain nitrotyrosine in position 10 and in positions 10 and 21, respectively. Both retain full trypsin inhibitor activity. Measurements of optical rotatory dispersion in the 230 nm region indicate that the secondary structure of the protein is unaffected by nitration. The nitrated derivatives exhibit a conformation-dependent side-chain Cotton effect in the region of nitroaromatic absorption. No such Cotton effect is shown by a nitrotyrosine-containing pentadecapeptide isolated from the tryptic digest from the nitrated and oxidized inhibitor. We have earlier shown [a] that the optical rotatory dispersion curve of pancreatic trypsin inhibitor shows a Cotton effect at 278 nm due to the tyrosine residues and that this effect is strongly dependent on conformation. It therefore appeared of interest to modify the tyrosine residues and to study the effect of such modification on the inhibitory activity and optical rotatory dispersion behaviour of pancreatic trypsin inhibitor. The recently introduced tetranitromethane reagent [9-111 has accordingly been used to nitrate pancreatic trypsin inhibitor and the properties of the nitrated product have been investigated. MATERIAL AND METHODSPancreatic trypsin inhibitor was prepared and characterized as described in the preceding paper [4].Non-Standard Abbreviation. Dansyl, I-dimethylaminonaphthalene-5-sulfonyl. Enzyme. Trypsin (EC 3.4.4.4).Trypsin used for the digestion was a crystalline preparation obtained from LBFiva (Prague) and was recrystallized three times with magnesium sulfate. Its residual chymotryptic activity was inhibited by reaction with l-chloro-4-phenyl-3-tosylamino-2-bu-The activity of trypsin was determined according to Nagel et al.[13] from the increase of absorbance at 405 nm due to the p-nitraniline liberated from benzoyl-DL-arginine p-nitranilide.Pancreatic trypsin inhibitor activity was determined by a difference method. The solution to be assayed (50 pl) was incubated with 100 pl of 0.1 M Tris-HC1 buffer, pH 7.8, and 250 pl of 0.001 N HC1 containing 40 pg of trypsin at 25" for 1, 5, I0 or 15 minutes and the activity of the remaining trypsin was determined as above.Protein concentrations were measured spectrophotometrically using A&ilo = 17.24 for trypsin s o htions and AiAlo = 8.33 for solutions of the inhibitor.Ultraviolet absorption spectra and optical rotatory dispersion curves were measured on a Jasco ORD/UV-5 recording spectrophotometer and spectropolarimeter. The absorption spectra were recorded in 1 cm cells. Solutions in 0.05 M sodium acetate (pH 4.6) and in 0.1 M Tris-HC1 buffer (pH 7.8) were prepared from weighed amounts of the dry protein (about 0.04 g/100 ml). Optical rotatory dispersion measurements were performed with the same solutions in cells of 1.0, 0.1, and 0.01 dm light path. For some optical rotatory dispersion measurements anhydrous trifluoroacetic acid served as the solvent. The so...

Corrigendum

“…The pKs of Tyr 10 and Tyr 21 are close to that of aqueous tyrosine (pK 10.1), whereas those of Tyr 23 and Tyr 35 deviate considerably. This and the preferential nitration 14 of Tyr 10 and Tyr 21 are as expected for freely accessible surface residues. Exhaustive iodination results in modification of Tyr 35 as well 15 .…”

On the Use of Tetranitromethane as a Nitration Reagent. The Reaction of Phenol Side-Chains in Bovine and Porcine Trypsinogens and Trypsins

“…4 has been found for the essential histidine 46 of the active center in the acylenzyme as compared to 6.5 for native trypsin in the same conditions.…”

o‐Nitrotyrosine andp‐iodophenylalanine as spectroscopic probes for structural characterization of SH3 complexes

“…Furthermore, the l max value of the newly generated band suggests that NT binds in the Trp pocket of Myo3-SH3 in its neutral -OH form, which maximally absorbs at 350 nm ( Fig. 3C; Meloun et al 1968). Similar results have been recently reported for the binding of NT analogs of hirudin to thrombin (De Filippis et al 2006).…”

“…The spectrum of free P2NT1 presents a marginally weak negative signal in the 300-390-nm range and a low-intensity positive band at 280 nm, assigned to the 1 A 1g ! 1 B 1u transition of NT (Meloun et al 1968;De Filippis et al 2006), which acquires some rotational strength because of the presence of several rigid prolines that stabilize the PP II conformation.…”