The reaction of tyrosyl residues of bovine trypsin and trypsinogen with tetranitromethane

Kenner, R. A.; Walsh, Kenneth A.; Neurath, Hans

doi:10.1016/0006-291x(68)90577-9

Cited by 28 publications

(9 citation statements)

References 12 publications

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“…Nitration was performed using a slightly modified method of Kenner et al [30]. To 1-2 ml 0.4 mM trypsin in buffer (50 mM Tris/HCI, 50 mM CaC12, pH 8.0, with or without 5 mM benzamidine), aliquots of 0.1 -2 M tetranitromethane in 96% ethanol were added (a 10-200 molar excess over trypsin).…”

Section: Nitration Of Tyrosine Residues Of Trypsinmentioning

confidence: 99%

“…Spectral investigations have shown [39] that four tyrosines are entirely exposed to the solvent and can undergo chemical modification more readily than the other tyrosine residues in trypsin. Kenner et al [30] showed that the treatment of trypsin with a moderate (10 -200-fold) excess of tetranitromethane specifically modifies these external four tyrosines; a much greater excess of the reagent must be used to make the internal tyrosines react. Tryptophan and other amino acid residues are marginally [30, 401 affected by tetranitromethane under these conditions.…”

Section: Nitration and Amination Of Tyrosine Residues In Trypsinmentioning

confidence: 99%

“…The next step, i.e. the reduction of nitrotyrosines, also proceeds specifically, and no other residues in the covalent structure of trypsin [30] or other enzymes [27] are affected as a result of treatment with sodium dithionite.…”

Section: Nitration and Amination Of Tyrosine Residues In Trypsinmentioning

confidence: 99%

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Protein stabilization via hydrophilization

Mozhaev

Šikšnis

Melik‐Nubarov

et al. 1988

European Journal of Biochemistry

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This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach.1. The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two-step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one.2. a-Chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxylic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of cr-chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll-400/Dextran-70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65 -98 "C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date.Applications of enzymatic catalysis in biotechnology, fine organic synthesis, analysis, medicine, and other areas are often hindered because many enzymes, if isolated from their natural environment in vivo, become unstable and rapidly inactivate (denature); for review, see [l, 21. For these reasons the problem of enzyme stabilization has received considerable attention in recent years [3 -81.The analysis of the structure-stability relationships in the protein leads us to the conclusion [S] that the structure of proteins is not optimal with regard to their stability. There are still some reverses for stabilization which are used by nature for the production of extremely stable proteins, such as, for example, enzymes from thermophilic microorganisms ; for review, see [S, 91. Reserves of such a kind may be useful for artificial stabilization of enzymes. We shall discuss here, from this viewpoint, how to improve the protein structure in order to make enzymes more stable.According to X-ray crystallographic data, about one half of the surface area of proteins is occupied by nonpolar amino Corre2pondence to K. Martinek, Ustav OrganickC Chemie a Biochemie, Ceskoslovenska Akademie Vtid, Flemingovo nimEsti 2, CS-166-10 Praha, CzechoslovakiaThe authors wish to dedicate this paper to the memory of their teacher and friend. Professor...

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Section: Nitration Of Tyrosine Residues Of Trypsinmentioning

confidence: 99%

Section: Nitration and Amination Of Tyrosine Residues In Trypsinmentioning

confidence: 99%

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Protein stabilization via hydrophilization

Mozhaev

Šikšnis

Melik‐Nubarov

et al. 1988

European Journal of Biochemistry

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“…We did not focus our attention on the localization of the surface or masked tyrosyl side-chains in the trypsin sequence. An independant work done by Kenner et al [9] has shown that tyrosines 11, 28, 48 and 137 were preferentially nitrated. This result is in agreement with the localization of masked phenols in trypsinogen which has been previously postulated by Delaage et al [12] to be tyrosines 20, 82, 158, 212.…”

mentioning

confidence: 97%

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

1970

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Tetranitromethane, a reagent described by Riordan et al. in 1966 has been used for the nitration of tyrosines in bovine and porcine trypsinogen and trypsin. The reagent induces a great number of inter-and intra-molecular side-reactions. I n particular, considerable polymerization due to crosslinking can be observed, especially with high concentrations of proteins. Monomers, dimers and polymers of higher degree can be easily separated by Sephadex G-100 chromatography. The existence of polymeric species has also been observed with ribonuclease, lysozyme and the Kunitz pancreatic inhibitor. 5 of the 10 tyrosines are sufficiently exposed a t the surface of the trypsinogen molecule to be nitrated easily. The second-order kinetics of the chemical modification have been studied under different conditions and compared to the kinetics of nitration of N-acetyl-L-tyrosine ethyl ester.Nitration increases the bulk of the tyrosine side-chain and decreases the pK-of the phenol function from 10.2-10.3 to 6.9 in trypsinogen. This important modification of the properties of this residue has no effect on the catalytic activity of trypsin toward small synthetic ester or amide substrates but it decreases considerably the tryptic activity toward macromolecular substrates. For example, nitration of trypsin decreases by a factor of 15 the maximal rate of activation of chymotrypsinogen A; no change on K , has been observed.Since its introduction in the protein field by Riordan et al. [1,2], tetranitromethane seems to be the most widely used reagent for a preferential modification of tyrosyl residues [3-111.The work presented here was undertaken in order to study the mechanism of action of tetranitromethane and to establish the functional and structural consequences of the nitration. We did not focus our attention on the localization of the surface or masked tyrosyl side-chains in the trypsin sequence. An independant work done by Kenner et al.[9] has shown that tyrosines 11, 28, 48 and 137 were preferentially nitrated. This result is in agreement with the localization of masked phenols in trypsinogen which has been previously postulated by Delaage et al. [12] to be tyrosines 20, 82, 158, 212. This paper will emphasize the dangers of using tetranitromethane for structure-function relationships studies without consideration of the numerous sidereactions which always occur in addition to the main one, the specific nitration of tyrosines. METHODS

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“…To explore whether this modification alters also the specificity towards polypeptides, our present study was aimed at the comparison of the specificity of cleavage of the chain B of insulin by OL-and 6-chymotrypsins and their nitrated forms. The results obtained with chymotrypsiis were compared with those obtained with its structural homologue, trypsin: In contrast with previous papers dealing with the nitrated heterogeneous trypsin [3,4] , a homogeneous monomeric nitrated /3-trypsin was isolated. Its properties differ partly from those observed previously.…”

Section: Introductionmentioning

confidence: 99%