scholarly journals SIMULTANEOUS HYDROLYSES OF ESTERS AND PROTEINS AT SATURATION LEVELS

1958 ◽  
Vol 41 (3) ◽  
pp. 485-500 ◽  
Author(s):  
M. Castañeda-Agulló ◽  
Luz M. Del Castillo
Keyword(s):  
Enzyme System ◽  
Kinetic Studies ◽  
Direct Titration ◽  
Enzyme Substrate ◽  
Constant Ph ◽  
Potentiometric Measurement ◽  
The Individual ◽  
Hydrolysis Of ◽  
Protein Enzyme

A direct titration method for the determination of proteolytic activity is discussed. This involves the potentiometric measurement of the volume of 0.08 N NaOH required to maintain a constant pH (8.0) during the time of the hydrolysis. It is a sensitive method which presents several advantages; viz., it measures simultaneously protease and esterase activity, it follows the hydrolysis very closely and from the first stages; the titration is continuous and on the same sample. This method determines a constant fraction of the groups titratable by formol titration. The ratio formol: direct titration is represented by a factor "f" which is presumed to be distinct for each protein-enzyme system. Kinetic studies, using this method, revealed that the rates of hydrolysis of mixtures casein-gelatin on one hand, casein-BAEE or gelatin-BAEE on the other, are always larger than those of the corresponding isolated substrates. In many cases the resulting rates are equal or nearly equal to the sum of the individual rates, even though the mentioned rates have been determined within the saturation zones for every substrate. The former observations are inconsistent with the theory of the formation of an intermediary enzyme-substrate compound, unless it is assumed that the enzyme has a specific active group for each substrate.

THE PATTERN OF URINARY EXCRETION OF INDIVIDUAL 17-KETOSTEROIDS IN RHESUS MONKEYS

1971 ◽  
Vol 67 (4) ◽  
pp. 711-720 ◽  
Author(s):  
S. B. Pal
Keyword(s):  
Rhesus Monkeys ◽  
Enzyme Hydrolysis ◽  
Colorimetric Determination ◽  
Day Range ◽  
Extraction Separation ◽  
Normal Mean ◽  
The Individual ◽  
Hydrolysis Of ◽  
Steroid Conjugates

ABSTRACT A systematic procedure for the extraction, separation, purification and colorimetric determination of urinary individual 17-ketosteroids in normal rhesus monkeys is described. Dehydroepiandrosterone was extracted after hot hydrolysis of the urine at neutral pH and the remaining steroid conjugates were extracted at pH 2 by the addition of ammonium sulphate. The liberated steroids were extracted after enzyme hydrolysis and the ketonic steroids were separated from the non-ketones by the Girard T reaction. The 11-deoxy steroids were separated from the 11-oxygenated steroids on a Celite column. They were further separated by paper chromatography and after elution, the individual 17-ketosteroids were estimated by the Zimmermann reaction. Normal mean excretion values in 30 female monkeys for total neutral 17-ketosteroids were 1.8 mg/24 h (range 1.5–2.6); total 17-hydroxycorticosteroids, 3.1 mg/24 h (range 2.2–4.8); androsterone, 38.6 μg/kg/24 h (range 9.3–60.2); aetiocholanolone, 19.2 μg/kg/24 h (range 6.8–30.7); dehydroepiandrosterone, 30.8 μg/kg/24 h (range 13.4–50.6); 11-ketoandrosterone, 7.5 μg/kg/24 h (range 5.1–14.1); 11-ketoaetiocholanolone, 10.2 μg/kg/24 h (range 6.8–18.3); 11β-hydroxyandrosterone, 15.4 μg/kg/24 h (range 8.8–26.7); 11β-hydroxyaetiocholanolone, 19.6 μg/kg/24 h (range 12.2–33.1). For total neutral 17-ketosteroids in 20 males, they were 2.5 mg/day (range 2.3–4.1); total 17-hydroxycorticosteroids, 3.7 mg/day (range 2.8–5.3); androsterone, 51.5 μg/kg/24 h (range 12.3–76.7); aetiocholanolone, 22.6 μg/kg/24 h (range 8.7–42.3); dehydroepiandrosterone, 41.3 μg/kg/24 h (range 16.9–59.7); 11-ketoandrosterone, 9.6 μg/kg/24 h (range 6.4–16.3); 11-ketoaetiocholanolone, 12.3 μg/kg/24 h (range 8.2–20.5); 11β-hydroxyandrosterone, 17.5 μg/kg/24 h (range 11.7–29.2); 11β-hydroxyaetiocholanolone, 21.7 μg/kg/24 h (range 14.5–36.4).

ESTIMATION OF CARBOXYL, ALDEHYDE, AND KETONE GROUPS IN CHROMIUM TRIOXIDE OXYSTARCHES

1953 ◽  
Vol 31 (9) ◽  
pp. 801-813 ◽  
Author(s):  
A. C. Ellington ◽  
C. B. Purves
Keyword(s):  
Corn Starch ◽  
Chromium Trioxide ◽  
Hydroxylamine Hydrochloride ◽  
Hydriodic Acid ◽  
Carbonyl Groups ◽  
Sodium Bisulphite ◽  
Direct Titration ◽  
Hydrolysis Of ◽  
Bromide Solutions

Under identical conditions, corn starch amylose was more readily oxidized than the amylopectin by chromium trioxide dissolved in acetic acid – acetic anhydride, which was a nonswelling system. These differences in rate nearly vanished for samples dried through solvent exchange when the oxidant was dissolved in a swelling medium, 0.2 M aqueous sulphuric acid. The absolute rate of oxidation, however, was greatly reduced.Carboxyl groups in the oxystarches were satisfactorily estimated either by ion-exchange with calcium acetate or sodium bromide solutions, or by direct titration to about pH 8.5 with aqueous alkali. The samples retained chromium compounds which grossly interfered with the determination of total carbonyl groups by condensation with hydroxylamine hydrochloride; condensation with excess sodium cyanide, and estimation of the ammonia from the hydrolysis of the cyanohydrins, gave better results.Aldehyde groups in an oxystarch containing 0.16 M. of carboxyl and 0.14 M. of carbonyl groups were selectively oxidized with chlorous acid or alkaline hypoiodite, or were selectively condensed with sodium bisulphite solution. All three estimations indicated that about one-third of the carbonyl groups were aldehydes probably occupying the sixth positions in the glucose residues. The cyanohydrin of the oxystarch, when saponified and then hydrolyzed and reduced with boiling hydriodic acid, yielded the lactone of 2-methyl-4-hydroxyhexanoic acid, the recovery of which showed that at least 17% of the carbonyl groups occurred as 2-ketoglucose residues.

Studies on Soil Reaction. VII. An Electrodialysis Apparatus for the Determination of Replaceable Bases in Soils

1931 ◽  
Vol 21 (3) ◽  
pp. 484-492 ◽  
Author(s):  
J. K. Basu
Keyword(s):  
Simple Form ◽  
Water Soluble ◽  
Soil Reaction ◽  
Soluble Salts ◽  
Direct Titration ◽  
Influence Of Water ◽  
Electrodialysis Cell ◽  
The Individual

1. A simple form of two-compartment electrodialysis cell is described for the determination of replaceable bases in sets of six soils at a time. The determination requires little attention and the total replaceable bases are obtained by direct titration.2. The disturbing influence of water-soluble salts is examined and it is shown that the technique may be modified so as to exclude the kations of soluble salts from either the total bases as determined by direct titration of the dialysate or from the individual bases as determined by analysis.

scholarly journals The linkage between binding of the C-terminal domain of hirudin and amidase activity in human α-thrombin

1993 ◽  
Vol 289 (2) ◽  
pp. 475-480 ◽  
Author(s):  
R de Cristofaro ◽  
B Rocca ◽  
B Bizzi ◽  
R Landolfi
Keyword(s):  
Rate Constants ◽  
Binding Constant ◽  
Amidase Activity ◽  
Equilibrium Binding ◽  
Viscosity Effects ◽  
The Individual ◽  
Individual Rate ◽  
Hydrolysis Of ◽  
Equilibrium Binding Constant

A method derived from the analysis of viscosity effects on the hydrolysis of the amide substrates D-phenylalanylpipecolyl-arginine-p-nitroaniline, tosylglycylprolylarginine-p-nitroanaline and cyclohexylglycylalanylarginine-p-nitroalanine by human alpha-thrombin was developed to dissect the Michaelis-Menten parameters Km and kcat into the individual rate constants of the binding, acylation and deacylation reactions. This method was used to analyse the effect of the C-terminal hirudin (residues 54-65) [hir-(54-65)] domain on the binding and hydrolysis of the three substrates. The results showed that the C-terminal hir-(54-65) fragment affects only the acylation rate, which is increased approx. 1.2-fold for all the substrates. Analysis of the dependence of acylation rate constants on hirudin-fragment concentration, allowed the determination of the equilibrium binding constant of C-terminal hir-(54-65) (Kd approximately 0.7 microM). In addition this peptide was found to competitively inhibit thrombin-fibrinogen interaction with a Ki which is in excellent agreement with the equilibrium constant derived from viscosity experiments. These results demonstrate that binding of hir-(54-65) to the fibrinogen recognition site of thrombin does not affect the equilibrium binding of amide substrates, but induces only a small increase in the acylation rate of the hydrolysis reaction.

scholarly journals Fluorogenic Substrates for the Determination of Plasminogen Activators and Plasmin

1977 ◽  
Author(s):  
W. Nieuwenhuizen ◽  
G. Wijngaards ◽  
E. Groeneveld
Keyword(s):  
Plasminogen Activator ◽  
Enzyme Histochemistry ◽  
Biological Fluids ◽  
Kinetic Studies ◽  
Plasminogen Activators ◽  
Fluorogenic Substrates ◽  
Highly Sensitive ◽  
Tissue Plasminogen ◽  
Hydrolysis Of

Plasminogen activator activities in biological fluids are low and specific for plasminogen. Highly sensitive and accurate assays are needed for direct quantification and for kinetic studies. We therefore, synthesized tripeptide amides i. e., t. BOC. val. gly. arg β-NA (l) and val. gly. arg β-NA (II) from which the fluorescent group β-naphtylamine (β-NA) is released upon enzymatic hydrolysis.Kinetic parameters found for the hydrolysis of these substrates by plasmin, urokinase and tissue plasminogen activator are:*in nmol/CU/min; **in pmol/CTA/minStrikingly, tissue activator is not active on substrate II, allowing discrimination between this activator and other proteases.The sensitivity of the assay is at least ten times higher than that observed for other synthetic substrates available. Moreover, substrates I and II can be used in enzyme histochemistry.

Cyclic voltammetric study of acid hydrolysis of tris(bipyridine) chromium(II): a critical appraisal of CV determination of kinetic parameters

1985 ◽  
Vol 63 (10) ◽  
pp. 2732-2735 ◽  
Author(s):  
Shachar D. Nadler ◽  
James G. Dick ◽  
Cooper H. Langford
Keyword(s):  
Critical Appraisal ◽  
Kinetic Studies ◽  
Cyclic Voltammetric ◽  
Residual Currents ◽  
Cyclic Voltammetric Study ◽  
Rate Determining Step ◽  
Voltammetric Study ◽  
Anodic Peak Current ◽  
Hydrolysis Of

Cyclic voltammetry has been exploited to reinvestigate discrepancies in the reported rate constant for the reaction:[Formula: see text]which follows electrode reduction of the substitution inert [Formula: see text]. It is shown that the problems arise from oversimplified treatment of residual currents when estimating the anodic peak current, ia. A "two scan" procedure for estimating the background currents is introduced which appears to resolve discrepancies. The kinetic studies were extended to a methyl-substituted bipyridine. Since ring methylation has very little effect on kf, it appears that the aquation reaction is initiated by a rate-determining step which requires a minimum of bipyridine rearrangement.

scholarly journals KPC-2 β-lactamase Enables Carbapenem Antibiotic Resistance Through Fast Deacylation of the Covalent Intermediate

2020 ◽  
pp. jbc.RA120.015050
Author(s):  
Shrenik C Mehta ◽  
Ian M Furey ◽  
Orville A Pemberton ◽  
David M Boragine ◽  
Yu Chen ◽  
...  
Keyword(s):  
Active Site ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Common ◽  
Covalent Intermediate ◽  
Hydrolysis Of ◽  
Carbapenem Antibiotic ◽  
Enzyme Intermediate

Serine active-site β-lactamases hydrolyze β-lactam antibiotics through formation of a covalent acyl-enzyme intermediate followed by deacylation via an activated water molecule. Carbapenem antibiotics are poorly hydrolyzed by most β-lactamases due to slow hydrolysis of the acyl-enzyme intermediate. However, the emergence of the KPC-2 carbapenemase has resulted in widespread resistance to these drugs, suggesting it operates more efficiently. Here, we investigated the unusual features of KPC-2 that enable this resistance. We show that KPC-2 has a 20,000-fold increased deacylation rate compared to the common TEM-1 β-lactamase. Further, kinetic analysis of active site alanine mutants indicates that carbapenem hydrolysis is a concerted effort involving multiple residues. Substitution of Asn170 greatly decreases the deacylation rate, but this residue is conserved in both KPC-2 and non-carbapenemase β-lactamases, suggesting it promotes carbapenem hydrolysis only in the context of KPC-2. X-ray structure determination of the N170A enzyme in complex with hydrolyzed imipenem suggests Asn170 may prevent the inactivation of the deacylating water by the 6α-hydroxyethyl substituent of carbapenems. In addition, the Thr235 residue, which interacts with the C3 carboxylate of carbapenems, also contributes strongly to the deacylation reaction. In contrast, mutation of the Arg220 and Thr237 residues decreases the acylation rate and, paradoxically, improves binding affinity for carbapenems. Thus, the role of these residues may be ground state destabilization of the enzyme-substrate complex or, alternatively, to ensure proper alignment of the substrate with key catalytic residues to facilitate acylation. These findings suggest modifications of the carbapenem scaffold to avoid hydrolysis by KPC-2 β-lactamase.

scholarly journals The effect of temperature on the individual stages of the hydrolysis of non-specific-p-nitrophenol esters by α-chymotrypsin

1977 ◽  
Vol 161 (1) ◽  
pp. 83-92 ◽  
Author(s):  
P A Adams ◽  
E R Swart
Keyword(s):  
Kinetic Studies ◽  
Detailed Comparison ◽  
Effect Of Temperature ◽  
Thermal Transitions ◽  
Arrhenius Plots ◽  
Ph Values ◽  
The Individual ◽  
Hydrolysis Of

Precise studies were performed on the effect of temperature on the rate and equilibrium parameters characterizing the individual stages of the alpha-chymotrypsin-catalysed hydrolysis of non-specific p-nitrophenol esters at pH 7.40 and 8.50. At both pH values the results indicate that a sharp kinetic anomaly is observed in Arrhenius plots of these parameters for the binding and acylation stages of the process, but not for the deacylation stage. Detailed comparison with other kinetic studies was made, and a comparison with thermal transitions observed in alpha-chymotrypsin by using physical techniques was attempted. A detailed discussion of possible causes of the anomalies is given.

Synthesis and Kinase Phosphorylation of 4-Deoxy-D-threohexulose

1973 ◽  
Vol 51 (7) ◽  
pp. 969-972 ◽  
Author(s):  
Clifford Raymond Haylock ◽  
Keith Norman Slessor
Keyword(s):  
Lithium Aluminum Hydride ◽  
Aluminum Hydride ◽  
Kinetic Studies ◽  
Lithium Aluminum ◽  
Substrate Complex ◽  
Acetobacter Suboxydans ◽  
Enzyme Substrate ◽  
Kinase Phosphorylation ◽  
Hydrolysis Of ◽  
Yeast Hexokinase

Synthesis of the only unknown deoxyfructose, 4-deoxy-D-threohexulose, is reported. Its preparation involved reductive lithium aluminum hydride ring opening of 3,4-anhydro-1,2:5,6-di-O-isopropylidene- D-talitol, followed by hydrolysis of the resulting epimeric deoxy diisopropylidene hexitols and selective Acetobacter suboxydans oxidation of 3-deoxy-D-arabinohexitol. Kinetic studies using 4-deoxy-D-threohexulose as substrate for yeast hexokinase support the premise that the C-4 hydroxyl is a binding group in formation of the enzyme–substrate complex. Enzymatic synthesis of 4-deoxy-D-threohexulose 6-phosphate and 4-deoxy-D-threohexulose 1,6-diphosphate has been achieved in low yield from 4-deoxy-D-threohexulose.

scholarly journals Obtaining and characteristic of the magnesium organic forms on the basis of products of bifidobacteria processing and their metabolites

2018 ◽  
Vol 12 (3) ◽  
Author(s):  
A. Kapustian ◽  
O. Antipina ◽  
R. Budiak
Keyword(s):  
Enzymatic Hydrolysis ◽  
Cell Walls ◽  
Bifidobacterium Bifidum ◽  
Mixed Ligand ◽  
Ultrasound Treatment ◽  
Bacterial Cells ◽  
Scanning Calorimetry ◽  
Enzyme Substrate ◽  
Hydrolysis Of

The possibility of obtaining bioavailable mixed ligand chelate complexes of Magnesium has been considered. As bioligands, it is proposed to use the metabolites and products of enzymatic hydrolysis of the peptidoglycans of the cell walls of Bifidobacterium bifidum AC-1670. As ligands, fragments of peptidoglycans of cell walls of bifidobacteria, which have their own immunotropic effects, were used. Destruction of bacterial cells was done by ultrasound treatment with subsequent enzymatic hydrolysis with papain. It was found that the highest content of potential ligands for chelation was obtained by ultrasound treatment at a frequency of 35 kg for 600 seconds with subsequent enzymatic hydrolisys, which lasted for 180 minutes at a ratio of the enzyme: substrate 1:1. In this case, the accumulation of amino acids in the hydrolyzate was 11.35 mg/cm3, low molecular weight peptides - 7.54 mg/cm3. The liquid phase of the product of the disintegration of the bacterial mass is investigated for the presence of metabolites that can participate in the formation of chelating magnesium complexes. Qualitative composition and quantitative content of organic acids are determined. It is established that in the product of disinfection of bifidobacteria the following acids are present: acetic (445.5 mg/dm3), lactic (284.6 mg/dm3), benzoic (1.3 mg/dm3). It has been established that the obtained mixed ligand systems are effective chelating agents and bind magnesium in an amount of 14 mg/cm3. The method of IR spectroscopy has proved that this system is formed with the participation of polydentant ligands. Determination of the pH stability of the complex showed that in the range of pH values 4–7, the chelate system is stable, at pH 2 only 10% of the complex is stored, at a pH of 9 – 60%. The thermostability of the complex was investigated by the method of differential scanning calorimetry. It was established that the complex is stable in the temperature range of 20-122 ° С, and therefore can be used as a physiologically functional ingredient in the health foods, the technology of which involves high-temperature processing.

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