Kinetics of the Reaction of Nitrous Acid with Model Compounds and Proteins, and the Conformational State of N-terminal Groups in the Chymotrypsin Family

1972 ◽  
Vol 50 (12) ◽  
pp. 1282-1296 ◽  
Author(s):  
A. Kurosky ◽  
T. Hofmann
Keyword(s):  
Nitrous Acid ◽  
Conformational State ◽  
Serine Proteinases ◽  
Amino Groups ◽  
Model Compounds ◽  
Third Order ◽  
Tyrosine Residues ◽  
Reactive Groups ◽  
Primary Amino ◽  
Kinetics Of

The kinetics of the reaction of nitrous acid at 4° and pH 4.0 with various amino acids, peptides, and proteins were studied. The reaction with isoleucine methyl ester was found to have a linear dependence on the square of the HONO concentration showing that N2O3 was the reactive species. Third order nitrosation rate constants of primary amino groups showed a correlation with their pK values. They were calculated for the concentration of the unprotonated species to give intrinsic reactivities. The rate of nitrosation of acetyltryptophan to give N-nitrosoacetyltryptophan was found to be a linear function of the nitrous acid concentration. This nitrosation therefore follows a different mechanism. The reaction of nitrous acid with tyrosine residues was examined by spectrophotometry. The reaction was negligible compared to that of other groups. Acetylhistidine and imidazole did not react. Reactivities for α-amino groups, ε-amino groups, and other residues in proteins were compared. The conformational state of the N-terminal residues in serine proteinases, as revealed from their reactivities, is discussed in detail. It is concluded that nitrous acid reacts preferentially with "surface" residues and is a useful tool for exploring conformational states of reactive groups in proteins, especially α-amino groups and indole rings.

scholarly journals Chemically modified nylons as supports for enzyme immobilization. Polyisonitrile-nylon

1974 ◽  
Vol 143 (3) ◽  
pp. 497-509 ◽  
Author(s):  
Leon Goldstein ◽  
Amihay Freeman ◽  
Mordechai Sokolovsky
Keyword(s):  
Aqueous Medium ◽  
Enzyme Immobilization ◽  
Functional Group ◽  
Carboxyl Groups ◽  
Partial Hydrolysis ◽  
Amino Groups ◽  
Tyrosine Residues ◽  
Chemically Modified ◽  
Reactive Groups ◽  
Hydrolysis Of

Four-component condensations between amine, carboxyl, isocyanide and aldehyde lead to the formation of N-substituted amides (Ugi, 1962). The present paper describes the use of such condensations for the introduction of chemically reactive groups on to the polyamide backbone of nylon. Polyisonitrile-nylon was synthesized by partial hydrolysis of nylon-6 powder, followed by resealing of the newly formed −CO2... NH2− pairs via a four-component condensation, by using acetaldehyde and 1,6-di-isocyanohexane. Polyisonitrile-nylon could also be converted into a diazotizable arylamino derivative, polyaminoaryl-nylon, by a four-component condensation by using a bifunctional amine, pp′-diaminodiphenylmethane, in the presence of an aldehyde and a carboxylate compound. The versatility of four-component condensations involving the isocyanide functional group of polyisonitrile-nylon allowed coupling of proteins, in an aqueous medium at neutral pH, through either their amino or carboxyl groups. Trypsin and papain were bound to polyisonitrile-nylon through their amino groups by a four-component condensation by using acetaldehyde and acetate; conversely, succinyl-(3-carboxypropionyl-)trypsin, pepsin and papain were coupled through their carboxyl groups in the presence of acetaldehyde and an amine (Tris). Diazotized polyaminoaryl-nylon could be utilized for the immobilization of papain, via the tyrosine residues of the enzyme.

The reaction with nitrous acid of the "active site" N-terminal isoleucine in chymotrypsin and derivatives

1970 ◽  
Vol 48 (6) ◽  
pp. 671-681 ◽  
Author(s):  
Joan W. Dixon ◽  
T. Hofmann
Keyword(s):  
Active Site ◽  
Rate Constants ◽  
Nitrous Acid ◽  
Amino Group ◽  
Amino Groups ◽  
Guanidinium Chloride ◽  
Third Order ◽  
Potential Activity ◽  
Chymotrypsin B ◽  
Nitrophenyl Ester

Bovine α-chymotrypsin, δ-chymotrypsin, homoarginine-δ-chymotrypsin, and bovine chymotrypsin B were inactivated by nitrous acid at pH 3.8–4.4 and 0°. The potential activity of bovine chymotrypsinogen A was not affected under these conditions. The inactivation rates as measured with the substrates α-N-acetyl-L-tyrosine ethyl ester, carbobenzoxyglycine p-nitrophenyl ester, and p-nitrophenyl acetate, and by 3H-di-isopropyl phosphorofluoridate incorporation were identical with the deamination rates of the amino group of the N-terminal isoleucine-16, but were slower than the deamination rates of the amino groups of the N-terminals half-cystine-1 and alanine-149. It is concluded that the deamination of isoleucine-16 is directly responsible for the inactivation. Third-order deamination rate constants of the N-terminal isoleucine-16 were measured and the following values (in min−1M−2) were obtained: α-chymotrypsin, 0.4–0.6; homoarginine-δ-chymotrypsin, 0.05; di-isopropyl phosphoryl-α-chymotrypsin, [Formula: see text]; tosyl-α-chymotrypsin, 0.05; chymotrypsin B, 0.3; α-chymotrypsin in guanidinium chloride, 30–50; homoarginine-δ-chymotrypsin in guanidinium chloride, > 20. The deamination rate constants for the model dipeptides isoleucylvaline and valylvaline are 40 and 46, respectively (Kurosky, A., and Hofmann, T.: to be published). A comparison shows that the constants for the dipeptides and the two chymotrypsins in guanidinium chloride are very close and are probably those of a fully exposed amino group. The much lower constants for the other enzymes and derivatives represent the varying degrees of accessibility of the amino group and show the usefulness of the reagent as a conformational probe. The results are fully compatible with the proposed structure of α-chymotrypsin (1) and the proposed function of the N-terminal isoleucine (2).

Measurement of proteolysis in cheese: relationship between phosphotungstic acid-soluble N fraction by Kjeldahl and 2,4,6- trinitrobenzenesulphonic acid-reactive groups in water-soluble N

1994 ◽  
Vol 61 (3) ◽  
pp. 437-440 ◽  
Author(s):  
Yvette Bouton ◽  
Remy Grappin
Keyword(s):  
Amino Acids ◽  
Free Amino Acids ◽  
Free Amino ◽  
Phosphotungstic Acid ◽  
Water Soluble ◽  
Hydroxyl Groups ◽  
Amino Groups ◽  
Reactive Groups ◽  
Primary Amino

Free amino groups produced during cheese ripening are used to indicate the extent of cheese proteolysis. Several studies have shown a high correlation between the level of free amino acids and the flavour of Gouda (Aston et al. 1983) or Comté (Grappin & Berdagué, 1989). Measurement of the level of free amino acids seems useful for the investigation of flavour chemistry in cheese (Lemieux et al. 1990). The determination of N fractions is often used to estimate the degree of proteolysis in cheese, but since this procedure is laborious and time consuming several attempts have been made to replace it by more rapid methods (Ardö & Meisel, 1991). Since its introduction by Satake et al. (1960), the 2,4,6-trinitrobenzenesulphonic acid (TNBS) method has been widely used for the determination of free amino groups. Because TNBS does not react with the imino groups of histidine and proline or the hydroxyl groups of tyrosine, serine or threonine, it has been accepted as a selective reagent for primary amino groups (Burger, 1974). Measurement of N by Kjeldahl in the phosphotungstic acid (PTA)–sulphuric acid extract (Gripon et al. 1975) estimates the N of free amino acids and low molecular mass peptides. The purpose of this study was to compare the TNBS and PTA-soluble N methods in order to find out whether the TNBS procedure can replace that of PTA-soluble N in the determination of a cheese proteolysis index.

The Reactivity of the N-terminal Isoleucine of α-Chymotrypsin as a Function of Ionic Strength

1971 ◽  
Vol 49 (5) ◽  
pp. 529-534 ◽  
Author(s):  
A. Kurosky ◽  
J. E. S. Graham ◽  
Joan W. Dixon ◽  
T. Hofmann
Keyword(s):  
Ionic Strength ◽  
Nitrous Acid ◽  
High Ionic Strength ◽  
Amino Group ◽  
Model Compounds ◽  
Third Order ◽  
X Ray ◽  
A Value ◽  
Group A ◽  
Low Ionic Strength

The reactivity of the α-amino group of isoleucine-16 of α-chymotrypsin towards nitrous acid at pH 4.0 and 0° is strongly dependent on ionic strength. Third-order deamination rate constants at low ionic strength (μ = 0.1 M) are 500–1000 times higher than those at high ionic strength (μ = 5.0 and 6.0 M) and are independent of the nature of the ions and the chymotrypsin concentration. Extrapolation to zero ionic strength of a plot of the logarithms of the constants against ionic strength leads to a value which is the same as that for the exposed α-amino group of the model compounds isoleucylvaline and valylvaline, and of the N-terminal isoleucine of pepsin. The deamination rate constant of the dipeptide valylvaline varies only two- to three-fold between ionic strength of 0.1 M and 6 M. The results suggest that the concept of a "buried" N-terminal as shown by X-ray analysis (carried out at pH 4.2 and ionic strength 9–11 M) requires modification; at low ionic strength (0.1 M) the reactivity of the N-terminal is only little below that of an exposed amino group, a fact which suggests that the amino group is much more available than shown by the X-ray analysis. The results are interpreted in terms of an effect of the ionic strength on the equilibrium between two conformational states of the enzyme.

Aldehyde gold clusters for molecular labeling

1995 ◽  
Vol 53 ◽  
pp. 858-859
Author(s):  
James F. Hainfeld ◽  
Frederic R. Furuya
Keyword(s):  
Covalent Bond ◽  
Aldehyde Group ◽  
Gold Clusters ◽  
Side Reactions ◽  
Amino Groups ◽  
Sodium Cyanoborohydride ◽  
Schiff’S Base ◽  
Protein Molecules ◽  
Hydroxysuccinimide Esters ◽  
Primary Amino

Glutaraldehyde is a useful tissue and molecular fixing reagents. The aldehyde moiety reacts mainly with primary amino groups to form a Schiff's base, which is reversible but reasonably stable at pH 7; a stable covalent bond may be formed by reduction with, e.g., sodium cyanoborohydride (Fig. 1). The bifunctional glutaraldehyde, (CHO-(CH2)3-CHO), successfully stabilizes protein molecules due to generally plentiful amines on their surface; bovine serum albumin has 60; 59 lysines + 1 α-amino. With some enzymes, catalytic activity after fixing is preserved; with respect to antigens, glutaraldehyde treatment can compromise their recognition by antibodies in some cases. Complicating the chemistry somewhat are the reported side reactions, where glutaraldehyde reacts with other amino acid side chains, cysteine, histidine, and tyrosine. It has also been reported that glutaraldehyde can polymerize in aqueous solution. Newer crosslinkers have been found that are more specific for the amino group, such as the N-hydroxysuccinimide esters, and are commonly preferred for forming conjugates. However, most of these linkers hydrolyze in solution, so that the activity is lost over several hours, whereas the aldehyde group is stable in solution, and may have an advantage of overall efficiency.

Purification of Penicillin Amidohydrolase, an Enzyme for Semisynthetic Procedures

1992 ◽  
Vol 57 (10) ◽  
pp. 2187-2191 ◽  
Author(s):  
Jiří Jiráček ◽  
Tomislav Barth ◽  
Jiří Velek ◽  
Ivo Bláha ◽  
Jan Pospíšek ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Amino Groups ◽  
Primary Amino

Penicillin amidohydrolase (EC 3.5.1.11.) is one of the few enzymes used successfully for deprotection of primary amino groups of semisynthetic peptides. The available material is usually contamined by endo- and exopeptidases. We managed to prepare the enzyme devoid of trypsin- and chymotrypsin-like activities using affinity chromatography with specific ligands: Gly-D-Phe-Phe-Tyr-Thr-Pro-Lys-Thr (the fF peptide) and Leu-Gly-Val-D-Arg-Arg-Gly-Phe (the rR peptide). For further purification of the enzyme affinity chromatography with N-phenylacetyl-D-tert-Leu as a ligand was used.

Kinetics of the Imidazolium Ring-Opening of mRNA 5'-cap Analogs in Aqueous Alkali

2006 ◽  
Vol 71 (4) ◽  
pp. 567-578 ◽  
Author(s):  
Alicja Stachelska ◽  
Zbigniew J. Wieczorek ◽  
Janusz Stępiński ◽  
Marzena Jankowska-Anyszka ◽  
Harri Lönnberg ◽  
...  
Keyword(s):  
Electrostatic Interaction ◽  
Ring Opening ◽  
Nucleophilic Attack ◽  
Amino Groups ◽  
Hydroxide Ion ◽  
Methyl Group ◽  
Structural Modifications ◽  
Alkyl Groups ◽  
Imidazolium Ring ◽  
Kinetics Of

Second-order rate constants for the hydroxide-ion-catalyzed imidazolium ring-opening of several mono- and dinucleosidic analogs of mRNA 5'-cap have been determined. Intramolecular stacking of the two nucleobases in the dinucleosidic analogs, m7GpppN (m7G = 7-methylguanosine, N = 5'-linked nucleoside), and electrostatic interaction between the N-alkylated imidazolium ring and phosphate moiety have been shown to shield the m7G moiety against the nucleophilic attack of hydroxide ion. In addition, the effect of methylation of the nucleobase amino groups and replacement of the 7-methyl group with other alkyl groups have been studied. The influence of all the structural modifications studied turned out to be modest, the cleavage rates of the most and least reactive analogs (with the exception of non-phosphorylated nucleosides) differing only by a factor of 5.

Evidence for general base catalysis by protic solvents in a kinetic study of alcoholyses and hydrolyses of 1-(phenoxycarbonyl)pyridinium ions under both solvolytic and non-solvolytic conditions

2009 ◽  
Vol 74 (1) ◽  
pp. 43-55 ◽  
Author(s):  
Dennis N. Kevill ◽  
Byoung-Chun Park ◽  
Jin Burm Kyong
Keyword(s):  
Second Order ◽  
Base Catalysis ◽  
Substitution Reactions ◽  
Third Order ◽  
Methoxy Derivative ◽  
First Order ◽  
General Base ◽  
Protic Solvents ◽  
Kinetics Of ◽  
Pyridinium Ions

The kinetics of nucleophilic substitution reactions of 1-(phenoxycarbonyl)pyridinium ions, prepared with the essentially non-nucleophilic/non-basic fluoroborate as the counterion, have been studied using up to 1.60 M methanol in acetonitrile as solvent and under solvolytic conditions in 2,2,2-trifluoroethan-1-ol (TFE) and its mixtures with water. Under the non- solvolytic conditions, the parent and three pyridine-ring-substituted derivatives were studied. Both second-order (first-order in methanol) and third-order (second-order in methanol) kinetic contributions were observed. In the solvolysis studies, since solvent ionizing power values were almost constant over the range of aqueous TFE studied, a Grunwald–Winstein equation treatment of the specific rates of solvolysis for the parent and the 4-methoxy derivative could be carried out in terms of variations in solvent nucleophilicity, and an appreciable sensitivity to changes in solvent nucleophilicity was found.

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