THE INFLUENCE OF pH ON THE HYDROLYSIS OF BENZOYLCHOLINE BY PSEUDOCHOLINESTERASE OF HUMAN PLASMA

1964 ◽  
Vol 42 (2) ◽  
pp. 161-168 ◽  
Author(s):  
W. Kalow
Keyword(s):  
Human Plasma ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Enzyme Molecule ◽  
Acid Base ◽  
Enzyme Substrate ◽  
Michaelis Constants ◽  
Automatic Titrator ◽  
Influence Of Ph ◽  
Hydrolysis Of

The study was performed with an automatic titrator and purified human pseudocholinesterase prepared from pooled plasma. The data obtained are compatible with the assumption that each enzyme molecule contains two binding sites for benzoylcholine which are unlike in their dependence on pH. Michaelis constants and maximum hydrolysis velocities were derived for each of the two binding sites, and acid–base dissociation constants of the enzyme substrate complexes were estimated.

scholarly journals The kinetics of hydrolysis of some extended N-aminoacyl-l-arginine methyl esters by human plasma kallikrein. Evidence for subsites S2 and S3

1982 ◽  
Vol 203 (1) ◽  
pp. 149-153 ◽  
Author(s):  
P R Levison ◽  
G Tomalin
Keyword(s):  
Human Plasma ◽  
Methyl Esters ◽  
Plasma Kallikrein ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Kinetics Of Hydrolysis ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Hydrolysis Of

Subsites in the S2-S4 region were identified in human plasma kallikrein. Kinetic constants (kcat., Km) were determined for a series of seven extended N-aminoacyl-L-arginine methyl esters based on the C-terminal sequence of bradykinin (-Pro-Phe-Arg) or (Gly)n-Arg. The rate-limiting step for the enzyme-catalysed reaction was found to be deacylation of the enzyme. It was possible to infer that hydrogen-bonded interactions occur between substrate and the S2-S4 region of kallikrein. Insertion of L-phenylalanine at residue P2 demonstrates that there is also a hydrophobic interaction with subsite S2, which stabilizes the enzyme-substrate complex. The strong interaction demonstrated between L-proline at residue P3 and subsite S3 is of greatest importance in the selectivity of human plasma kallikrein. The purification of kallikrein from Cohn fraction IV of human plasma is described making use of endogenous Factor XIIf to activate the prekallikrein. Kallikreins I (Mr 91 000) and II (Mr 85 000) were purified 170- and 110-fold respectively. Kallikrein I was used for the kinetic work.

Influence Of Fibrin And Fibrinogen On The Activation Of Plasminogen By Urokinase

1981 ◽  
Author(s):  
Y Takada ◽  
A Takada
Keyword(s):  
Enzymatic Activity ◽  
Human Plasma ◽  
Binding Sites ◽  
Degradation Products ◽  
Feedback Mechanism ◽  
Plasmin Activity ◽  
Activation Rate ◽  
Extensive Degradation ◽  
Early Degradation ◽  
Hydrolysis Of

Human plasma was clotted with thrombin or Ca++ in the presence of urokinase (UK), and the activity of plasmin was measured by hydrolysis of S-2251. As a control, the hydrolysis of S-2251 was measured without the addition of thrombin (in the absence of clot). The hydrolysis of S-2251 was higher in the clot than in the plasma. When euglobulin solution was clotted by thrombin in the presence of UK, the hydrolysis of S-2251 was higher in the presence than in the absence of clot. Thus the increased hydrolysis of S-2251 in the clot after the addition of UK was not due to the interaction of plasmin with inhibitors (∝2AP or∝2M) in the presence of clot, but plasminogen (plg) seems to be better activated by UK in the presence of fibrin. When purified Glu-plg was used, Glu-plg was better activated by UK in the presence of fibrin than fibrinogen. The addition of AP to the mixture of Glu-plg and UK resulted in complete inhibition of plasmin activity but the presence of fibrin partially prevented its inactivation by ∝2AP. When Glu-plg was incubated with plasmin,resuiting Lys-plg was far better activated by UK than Glu-plg, but the presence of fibrin slightly enhanced its activation rate. When fibrinogen or fibrin was degraded by plasmin, early FDP enhanced the activation more than early FgDP. Early FgDP still better activates Glu-plg than fibrinogen. Extensive degradation products (D and E) resulted in less activation of Glu-plg than in the absence of these products. The addition of these degradation products to already activated plasmin had no effect on the enzymatic activity of plasmin.In conclusion, Glu-plg was better activated by UK when clot was formed. Binding of Glu-plg with fibrin through lysine binding sites protected it from inactivation by AP. Early degradation also resulted in better activation of Glu-plg, but extensive degradation resulted in less activation which may be an important feedback mechanism of fibrinolysis.

scholarly journals The Role of Evolving Interfacial Substrate Properties on Heterogeneous Cellulose Hydrolysis Kinetics

2019 ◽  
Author(s):  
Jennifer Nill ◽  
Tina Jeoh
Keyword(s):  
Binding Sites ◽  
Binding Capacity ◽  
Cellulose Hydrolysis ◽  
Reaction Rates ◽  
Hydrolysis Kinetics ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Substrate Properties ◽  
Hydrolysis Of

AbstractInterfacial enzyme reactions require formation of an enzyme-substrate complex at the surface of a heterogeneous substrate, but often multiple modes of enzyme binding and types of binding sites complicate analysis of their kinetics. Excess of heterogeneous substrate is often used as a justification to model the substrate as unchanging; but using the study of the enzymatic hydrolysis of insoluble cellulose as an example, we argue that reaction rates are dependent on evolving substrate interfacial properties. We hypothesize that the relative abundance of binding sites on cellulose where hydrolysis can occur (productive binding sites) and binding sites where hydrolysis cannot be initiated or is inhibited (non-productive binding sites) contribute to rate limitations. We show that the initial total number of productive binding sites (the productive binding capacity) determines the magnitude of the initial burst phase of cellulose hydrolysis, while productive binding site depletion explains overall hydrolysis kinetics. Furthermore, we show that irreversibly bound surface enzymes contribute to the depletion of productive binding sites. Our model shows that increasing the ratio of productive- to non-productive binding sites promotes hydrolysis, while maintaining an elevated productive binding capacity throughout conversion is key to preventing hydrolysis slowdown.

Homogeneous substrate-labeled fluorescent immunoassay for theophylline in serum.

1981 ◽  
Vol 27 (1) ◽  
pp. 22-26 ◽  
Author(s):  
T M Li ◽  
J L Benovic ◽  
R T Buckler ◽  
J F Burd
Keyword(s):  
Binding Sites ◽  
Clinical Samples ◽  
Fluorescent Product ◽  
Theophylline Concentration ◽  
Enzyme Substrate ◽  
Competitive Protein Binding ◽  
Set Up ◽  
Hydrolysis Of ◽  
Beta Galactosidase ◽  
Control Samples

Abstract A substrate-labeled fluorescent immunoassay for theophylline in serum is described. 8-(3-Aminopropyl)-theophylline is covalently attached to a fluorogenic enzyme substrate, 7-beta-galactosylcoumarin-3-carboxylic acid. Hydrolysis of this theophylline-labeled substrate by beta-galactosidase yields a fluorescent product. When antibody to theophylline interacts with this substrate, the resulting complex is inactive as an enzyme substrate. For measuring theophylline, competitive protein-binding reactions are set up, with the theophylline in the sample competing with the substrate for the antibody-binding sites. The substrate not bound to antibody is hydrolyzed by beta-galactosidase, producing fluorescence that is proportional to the theophylline concentration. Results for theophylline determined by this method in clinical samples of serum correlated well (r > 0.96) with results obtained by gas-chromatographic or enzyme immunoassay procedures. The within-run CV for three control samples ranged from 1.1 to 2.8%, the between-run CB from 2.3 to 4.5%.

Influence of Coagulation on the Activation of Plasminogen by Streptokinase and Urokinase

1979 ◽  
Vol 42 (03) ◽  
pp. 901-908 ◽  
Author(s):  
Akikazu Takada ◽  
Tetsumei Urano ◽  
Yumiko Takada
Keyword(s):  
Human Plasma ◽  
Fibrin Clot ◽  
Chromogenic Substrate ◽  
Hydrolysis Of

SummaryHuman plasma was mixed with Ca++ or thrombin, and urokinase (UK) or streptokinase (SK) and a chromogenic substrate (S-2251: H-D-Val-Leu-Lys-pNA) specific to plasmin. The hydrolysis of S-2251 was higher when clot was formed by the addition of Ca++ or thrombin than in the absence of clot. The hydrolysis of S-2251 by euglobulin in the presence of UK was also higher when clot was formed, thus, inhibitors may not be related to the better activation of plasminogen, in the presence of fibrin clot. It may be suggested that plasminogen was better activated by activators (UK and SK) in the clot than in its absence.

Acid-base properties of 1,3-diphenyl-5-(1H-tetrazol-2-yl)formazan and 1,3-diphenyl-5-(2H-1,2,4-triazol-3-yl)formazan

1982 ◽  
Vol 47 (11) ◽  
pp. 2882-2889
Author(s):  
Nadezhda Likhareva ◽  
Ladislav Šůcha ◽  
Miloslav Suchánek
Keyword(s):  
Computer Program ◽  
Dissociation Constants ◽  
Acid Base ◽  
New Compounds ◽  
Base Properties

Two new compounds from the formazan series, viz. 1,3-diphenyl-5-(1H-tetrazol-2-yl)formazan and 1,3-diphenyl-5-(2H-1,2,4-triazol-3-yl)formazan, were prepared, and the dissociation constants and molar absorptivities of all of their acid-base species were determined spectrophotometrically employing the SPEKTFOT computer program.

Total weak acid concentration and effective dissociation constant of nonvolatile buffers in human plasma

2001 ◽  
Vol 91 (3) ◽  
pp. 1364-1371 ◽  
Author(s):  
Peter D. Constable
Keyword(s):  
Dissociation Constant ◽  
Human Plasma ◽  
Total Concentration ◽  
Weak Acid ◽  
Strong Ion Difference ◽  
Acid Base ◽  
Horse Plasma ◽  
Using Data ◽  
Species Specific

The strong ion approach provides a quantitative physicochemical method for describing the mechanism for an acid-base disturbance. The approach requires species-specific values for the total concentration of plasma nonvolatile buffers (Atot) and the effective dissociation constant for plasma nonvolatile buffers ( K a), but these values have not been determined for human plasma. Accordingly, the purpose of this study was to calculate accurate Atot and K a values using data obtained from in vitro strong ion titration and CO2tonometry. The calculated values for Atot (24.1 mmol/l) and K a (1.05 × 10−7) were significantly ( P < 0.05) different from the experimentally determined values for horse plasma and differed from the empirically assumed values for human plasma (Atot = 19.0 meq/l and K a = 3.0 × 10−7). The derivatives of pH with respect to the three independent variables [strong ion difference (SID), Pco 2, and Atot] of the strong ion approach were calculated as follows: [Formula: see text] [Formula: see text], [Formula: see text]where S is solubility of CO2 in plasma. The derivatives provide a useful method for calculating the effect of independent changes in SID+, Pco 2, and Atot on plasma pH. The calculated values for Atot and K a should facilitate application of the strong ion approach to acid-base disturbances in humans.

scholarly journals A spin label study of the thyroid hormone-binding sites in human plasma thyroxine transport proteins.

1981 ◽  
Vol 256 (2) ◽  
pp. 831-836
Author(s):  
S.Y. Cheng ◽  
G. Rakhit ◽  
F. Erard ◽  
J. Robbins ◽  
C.F. Chignell
Keyword(s):  
Thyroid Hormone ◽  
Human Plasma ◽  
Spin Label ◽  
Binding Sites ◽  
Transport Proteins ◽  
Hormone Binding ◽  
Label Study
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