scholarly journals Spectrophotometric measurement of the liberation of the α-amino group of cysteine residues in polypeptides

1966 ◽  
Vol 100 (2) ◽  
pp. 309-314 ◽  
Author(s):  
S Streichman ◽  
Y Avi-Dor
Keyword(s):  
Spectral Characteristics ◽  
Cysteine Residues ◽  
Amino Group ◽  
Peptide Bonds ◽  
Spectral Changes ◽  
Sh Group ◽  
Induced Changes ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Specific Peptide

1. The reaction between beta-bromopyruvic acid and SH groups of cysteine residues in reduced ribonuclease and in some other polypeptides was investigated. 2. One molecule of the acid was found to be necessary to block one SH group in reduced ribonuclease. The stoicheiometry of the interaction and the spectral characteristics of the compound formed suggested that the product is and S-oxalomethyl (R.S.CH(2).CO.CO(2)H) derivative of reduced ribonuclease. 3. Digestion of reduced S-oxalomethylated ribonuclease by trypsin or chymotrypsin induced changes in the spectrum that could be attributed to the liberation of the alpha-amino group of S-oxalomethylated cysteine residues from peptide bonds. The spectral changes that accompanied the hydrolysis of specific peptide bonds in reduced S-oxalomethylated ribonuclease and S-oxalomethylated co-poly(l-Lys,l-CySH) allowed the kinetics of the digestion to be followed. 4. Possible applications of the spectrophotometric method in the study of protein structure are discussed.

scholarly journals Proteolysis of β-lactoglobulin by Trypsin: Simulation by Two-Step Model and Experimental Verification by Intrinsic Tryptophan Fluorescence

Symmetry ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 153 ◽  
Author(s):  
M.M. Vorob’ev
Keyword(s):  
Tryptophan Fluorescence ◽  
Degree Of Hydrolysis ◽  
Peptide Bonds ◽  
Protein Substrates ◽  
Intrinsic Tryptophan Fluorescence ◽  
Step Model ◽  
Fluorescence Change ◽  
Β Lactoglobulin ◽  
Kinetics Of ◽  
Hydrolysis Of

To distinguish differences in enzymatic hydrolysis of various proteins, we propose an algorithm using a dataset of fluorescence spectra obtained at different moments of hydrolysis t. This algorithm was demonstrated in the example of β-lactoglobulin (β-LG) proteolysis by trypsin. The procedure involved processing the spectra to obtain the wavelength of the maximum fluorescence λmax, which was found to be proportional to the fraction of tryptophanes in hydrated proteolysis products (demasked tryptophanes). Then, the dependence λmax(t) was fitted by biexponential function with two exponential terms, one of which was responsible for the fast part of the fluorescence change during proteolysis. The contribution of this term was quite different for various protein substrates—it was positive for β-LG and negative for β-casein. The observed differences in proteolysis of different substrates were explained by different demasking processes. Combining the fluorescence data with the degrees of hydrolysis of peptide bonds allowed us to analyze the hydrolysis of β-LG in the framework of the two-step proteolysis model and estimate the ratio of rate constants of demasking and hydrolysis and the percentages of initially masked and resistant peptide bonds. This model predicted the existence of a bimodal demasking process with a fast part at the beginning of proteolysis and lag-type kinetics of release for some peptides. Compared with monitoring proteolysis in terms of the degree of hydrolysis only, the fluorescence data are helpful for the recognition of proteolysis patterns.

scholarly journals The reaction of 2,4,6-trinitrobenzenesulphonic acid with amino acids, peptides and proteins

1968 ◽  
Vol 108 (3) ◽  
pp. 383-391 ◽  
Author(s):  
R. B. Freedman ◽  
G. K. Radda
Keyword(s):  
Amino Acids ◽  
Glutamate Dehydrogenase ◽  
Free Amino ◽  
Reactive Species ◽  
Second Order ◽  
Amino Group ◽  
Exponential Rate ◽  
Amino Groups ◽  
Sh Group ◽  
Kinetics Of

1. The kinetics of the reaction of 2,4,6-trinitrobenzenesulphonic acid with various amino acids, peptides and proteins were studied by spectrophotometry. 2. The reaction of the α- and ∈-amino groups in simple amino acids was found to be second-order, and the unprotonated amino group was shown to be the reactive species. 3. By allowing for the concentration of unreactive −NH3+ group, intrinsic reactivities for the free amino groups were derived and shown to be correlated with the basicities. 4. The SH group of N-acetylcysteine was found to be more reactive to 2,4,6-trinitrobenzenesulphonic acid than most amino groups. 5. The reactions of insulin, chymotrypsinogen and ribonuclease with 2,4,6-trinitrobenzenesulphonic acid were analysed in terms of three exponential rate curves, each referring to one or more amino groups of the proteins. 6. The reaction of lysozyme with 2,4,6-trinitrobenzenesulphonic acid was found to display an acceleration effect. 7. From the reaction of 2,4,6-trinitrobenzenesulphonic acid with glutamate dehydrogenase at several enzyme concentrations, it was possible to discern two sets of amino groups of different reactivity, and to show that the number of groups in each set was decreased by aggregation of the enzyme.

The Steady State Kinetics of Plasmin and Trypsin Catalysed Hydrolysis of a Number of Tripeptideanilides

1979 ◽  
Author(s):  
U. Christensen ◽  
H-H. Ipsen
Keyword(s):  
Steady State ◽  
Kinetic Parameters ◽  
Amino Group ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Ph Range ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
State Kinetic

The steady state kinetic parameters of plasmin and trypsin catalysed hydrolysis of Bz-L-Phe-Val-Arg-pNA, L-Phe-Val-Arg-pNA, Bz-D-Phe-Val-Arg-pNA, D-Phe-Val-Arg-pNA and D-Val-Leu-Lys-pNA in the pH-range 6-9 are presented. Ionization of catalytically essential enzymic groups accounts satisfactorily for the pH-dependencies of the kinetic parameters for plas-rain and trypsin reactions with Bz-L-Phe-Val-Arg-pNA, Bz-D-Phe-Val-Arg-pNA and D-Val-Leu-Lys-pNA. The protonation of the α-amino group of L-Phe-Val-Arg-pNA and D-Phe-Val-Arg-pNA (pK=7.0) show some additional effect. The values of the catalytic constants for plasmin and trypsin reactions with these p-nitroanilides are alike those normally found for specific ester substrates, indicating that the deacylation steps are rate determining.

Fine Structural Localization of Acyltransferases Involved in Lipid Synthesis in Intestinal Mucosa.

1970 ◽  
Vol 28 ◽  
pp. 54-55
Author(s):  
R. J. Barrnett ◽  
J. A. Higgins
Keyword(s):  
Smooth Endoplasmic Reticulum ◽  
Lipid Synthesis ◽  
Mucosal Cell ◽  
Structural Localization ◽  
Morphological Studies ◽  
Mucosal Cells ◽  
Initial Appearance ◽  
Sh Group ◽  
Hydrolysis Of ◽  
Intestinal Mucosal Cells

The main products of intestinal hydrolysis of dietary triglycerides are free fatty acids and monoglycerides. These form micelles from which the lipids are absorbed across the mucosal cell brush border. Biochemical studies have indicated that intestinal mucosal cells possess a triglyceride synthesising system, which uses monoglyceride directly as an acylacceptor as well as the system found in other tissues in which alphaglycerophosphate is the acylacceptor. The former pathway is used preferentially for the resynthesis of triglyceride from absorbed lipid, while the latter is used mainly for phospholipid synthesis. Both lipids are incorporated into chylomicrons. Morphological studies have shown that during fat absorption there is an initial appearance of fat droplets within the cisternae of the smooth endoplasmic reticulum and that these subsequently accumulate in the golgi elements from which they are released at the lateral borders of the cell as chylomicrons.We have recently developed several methods for the fine structural localization of acyltransferases dependent on the precipitation, in an electron dense form, of CoA released during the transfer of the acyl group to an acceptor, and have now applied these methods to a study of the fine structural localization of the enzymes involved in chylomicron lipid biosynthesis. These methods are based on the reduction of ferricyanide ions by the free SH group of CoA.

Influence of medium on alkaline hydrolysis of succinic acid monomethyl and monopropyl esters

1980 ◽  
Vol 45 (11) ◽  
pp. 2873-2882
Author(s):  
Vladislav Holba ◽  
Ján Benko
Keyword(s):  
Succinic Acid ◽  
Kinetic Parameters ◽  
Alkaline Hydrolysis ◽  
Specific Interactions ◽  
Nonaqueous Media ◽  
The Media ◽  
Kinetics Of ◽  
Hydrolysis Of

The kinetics of alkaline hydrolysis of succinic acid monomethyl and monopropyl esters were studied in mixed aqueous-nonaqueous media at various temperatures and ionic strengths. The results of measurements are discussed in terms of electrostatic and specific interactions between the reactants and other components of the reaction mixture. The kinetic parameters in the media under study are related to the influence of the cosolvent on the solvation sphere of the reactants.

Belousov–Zhabotinskii type oscillation reaction with glycerol and kinetics of reactions between the system components

1987 ◽  
Vol 52 (10) ◽  
pp. 2375-2382 ◽  
Author(s):  
Ľubica Adamčíková ◽  
Peter Ševčík
Keyword(s):  
Oscillation System ◽  
Chemical Oscillations ◽  
Oscillation Reaction ◽  
Reaction Solution ◽  
System Components ◽  
Probable Source ◽  
Belousov Zhabotinskii Reaction ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Type Oscillation

Glycerol causes chemical oscillations in Belousov-Zhabotinskii reaction in a closed system as well as in a reaction solution bubbled with nitrogen. Since the oxidation of glycerol with bromate ions does not proceed autocatalytically and bromine in the oxidation state 0 or +1 in the absence of light does not react with glycerol, hydrolysis of bromine is the probable source of bromide ions in the studied oscillation system.

Kinetics of hydrolysis of peroxodisulphate ions in acidic medium to peroxomonosulphuric acid

1981 ◽  
Vol 46 (5) ◽  
pp. 1229-1236 ◽  
Author(s):  
Jan Balej ◽  
Milada Thumová
Keyword(s):  
Ionic Strength ◽  
Rate Constants ◽  
Acidic Medium ◽  
Measured Data ◽  
Molar Ratios ◽  
Starting Solution ◽  
Kinetics Of Hydrolysis ◽  
Rate Of Hydrolysis ◽  
Kinetics Of ◽  
Hydrolysis Of

The rate of hydrolysis of S2O82- ions in acidic medium to peroxomonosulphuric acid was measured at 20 and 30 °C. The composition of the starting solution corresponded to the anolyte flowing out from an electrolyser for production of this acid or its ammonium salt at various degrees of conversion and starting molar ratios of sulphuric acid to ammonium sulphate. The measured data served to calculate the rate constants at both temperatures on the basis of the earlier proposed mechanism of the hydrolysis, and their dependence on the ionic strength was studied.

Cyclization and acid-catalyzed hydrolysis of O-benzoylbenzamidoximes

1986 ◽  
Vol 51 (12) ◽  
pp. 2786-2797
Author(s):  
František Grambal ◽  
Jan Lasovský
Keyword(s):  
Reaction Rate ◽  
Kinetic Isotope ◽  
Acid Base Equilibrium ◽  
Mineral Acids ◽  
Kinetics Of Formation ◽  
Acid Catalyzed ◽  
Ph Scale ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Derivatives Of

Kinetics of formation of 1,2,4-oxadiazoles from 24 substitution derivatives of O-benzoylbenzamidoxime have been studied in sulphuric acid and aqueous ethanol media. It has been found that this medium requires introduction of the Hammett H0 function instead of the pH scale beginning as low as from 0.1% solutions of mineral acids. Effects of the acid concentration, ionic strength, and temperature on the reaction rate and on the kinetic isotope effect have been followed. From these dependences and from polar effects of substituents it was concluded that along with the cyclization to 1,2,4-oxadiazoles there proceeds hydrolysis to benzamidoxime and benzoic acid. The reaction is thermodynamically controlled by the acid-base equilibrium of the O-benzylated benzamidoximes.

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