scholarly journals A kinetic method for the study of solvent environments of thiol groups in proteins involving the use of a pair of isomeric reactivity probes and a differential solvent effect. Investigation of the active centre of ficin by using 2,2'- and 4,4'- dipyridyl disulphides as reactivity probes

1980 ◽  
Vol 185 (1) ◽  
pp. 217-222 ◽  
Author(s):  
J P G Malthouse ◽  
K Brocklehurst
Keyword(s):  
Dielectric Constant ◽  
Physicochemical Properties ◽  
Solvent Effect ◽  
Rate Constants ◽  
Kinetic Method ◽  
Thiol Group ◽  
Analogous Reaction ◽  
Thiol Groups ◽  
Active Centre ◽  
Effect Of Solvent

1. Whereas the second-order rate constants for the reaction of the thiolate ion of 2-mercaptoethanol with 4,4'-dipyridyl disulphide (k4PDS) and with 5,5'-dithiobis-2-nitrobenzoate dianion increase with decreasing dielectric constant of the solvent, or remain unchanged, the rate constant for the analogous reaction with 2,2'-dipyridyl disulphide (k2PDS) decreases. This anomalous solvent effect and other unusual physicochemical properties of 2,2'-dipyridyl disulphide are discussed. 2. The differential effect of solvent on the reactions of thiolate ion with the 2,2'- and 4,4'-dipyridyl disulphides is shown to provide a method of characterizing solvent environments of thiol groups in proteins by a reactivity-probe method that should not suffer from the usual drawback associated with the existence of steric or binding effects of unknown magnitude. Application of the method to ficin (EC 3.4.22.3) suggests that its active-centre thiol group resides in a relatively hydrophobic environment. 3. The pH-k profile for the reaction of ficin with 4,4'-dipyridyl disulphide is reported.

scholarly journals Effect of selective thiol-group derivatization on enzyme kinetics of (R)-3-hydroxybutyrate dehydrogenase

1993 ◽  
Vol 296 (3) ◽  
pp. 563-569 ◽  
Author(s):  
L A Dalton ◽  
J O McIntyre ◽  
S Fleischer
Keyword(s):  
Enzyme Kinetics ◽  
Kinetic Parameters ◽  
Thiol Group ◽  
Enzymic Activity ◽  
Reduction Product ◽  
Thiol Groups ◽  
Active Centre ◽  
Fold Reduction ◽  
Kinetics Of

(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a phosphatidylcholine-requiring tetrameric enzyme with two thiol groups (SH-1 and SH-2) per protomer. By first protecting the more rapidly reacting thiol group (SH-1) with diamide [1,1′-azobis-(NN′-dimethylformamide), DM] to form DM(SH-1)BDH, SH-2 can be selectively derivatized by reaction with maleimide reagents such as 4-maleimido-2,2,6,6-tetramethyl-piperidine-N-oxyl (MSL), which gives DM(SH-1)MSL(SH-2)BDH. Reduction with dithiothreitol (DTT) regenerates SH-1, yielding MAL(SH-2)BDH (where MAL is the diamagnetic reduction product of MSL-BDH and DTT). The enzymic activity of DM(SH-1)BDH is decreased to approx. 4% relative to the native purified enzyme, and the apparent Km for substrate, KmBOH, is increased approx. 100-fold. Reduction of DM(SH-1)BDH with DTT regenerates SH-1 and restores normal enzymic function. Modification of SH-2 with piperidinylmaleimide [MAL(SH-2)BDH] diminishes enzymic activity to approx. 35% of its original value, but has no significant effect on apparent KmBOH. The doubly derivatized enzyme, DM(SH-1)MSL(SH-2)BDH, has lower enzymic activity [about half that for DM(SH-2)BDH] and a yet higher apparent KmBOH than DM(SH-1)BDH. Derivatization of SH-2 with different maleimide reagents results in diminished activity approximately proportional to the size of the maleimide substituent, suggesting that this inhibition is steric. Whereas modification of SH-1 results in marked changes in kinetic parameters (increased apparent Km and reduced apparent Vmax), derivatization of SH-2 has a lesser effect on enzymic function. Thus SH-1 is postulated to be closer to the active centre than is SH-2, although neither is involved in catalysis, since: (1) the activity of the derivatized enzyme is not abolished; and (2) activity can be enhanced by increasing substrate (and cofactor) concentrations.

The Solvent Effects on Kinetics and Mechanism of Zinc or Cadmium Halide Catalyzed Reactions of Hydrosilanes with Hydroxylic Reagents

2004 ◽  
Vol 69 (12) ◽  
pp. 2281-2296 ◽  
Author(s):  
Jerzy J. Chruściel
Keyword(s):  
Dielectric Constant ◽  
Solvent Effects ◽  
Kinetic Method ◽  
Kinetic Studies ◽  
Reaction Rates ◽  
Nucleophilic Catalysis ◽  
Effect Of Solvent ◽  
Cadmium Bromide ◽  
Catalyzed Reactions

The effect of solvent composition on reaction rates was studied by a kinetic method for reactions of Et3SiH with Ph2Si(OH)2 in the presence of ZnCl2 in a mixture of DMF and THF or Et3SiH with PhCOOH and cadmium bromide CdBr2 in DMF-1,4-dioxane and DMF-THF. A decrease in reaction rates with decreasing polarity of the medium (and its dielectric constant) has been observed. The kinetic order of dehydrocoupling reaction with respect to DMF was determined from kinetic studies. The constants of nucleophilic catalysis with DMF (λ) were found. The role of DMF in the mechanism of dehydrocoupling of hydrosilanes with hydroxylic reagents in the presence of ZnX2 and CdX2 halides (X = Cl, Br, I) is discussed.

scholarly journals Characterization of the papain active centre by using two-protonic-state electrophiles as reactivity probes. Evidence for nucleophilic reactivity in the un-interrupted cysteine-25-histidine-159 interactive system

1978 ◽  
Vol 171 (2) ◽  
pp. 385-401 ◽  
Author(s):  
M Shipton ◽  
K Brochlehurst
Keyword(s):  
Thiol Group ◽  
Interactive System ◽  
Pyridyl Ring ◽  
Thiol Groups ◽  
Sulphur Atom ◽  
Active Centre ◽  
Nucleophilic Reactivity ◽  
Imidazolium Ion ◽  
Active Centres

1.2,2′-Dipyridyl disulphide (2-Py-S-S-2-Py) and n-propyl 2-pyridyl disulphide (propyl-S-S-2-Py) were used as two-protonic-state reactivity probes to investigate the active centre of papain (EC 3.4.22.2).2. The existence of a striking rate optimum at pH approx. 4 in the reaction of papain not only with the symmetrical probe but also with the unsymmetrical probe is shown to constitute compelling evidence that the thiolate ion component of the cysteine-25-histidine-159 interactive system of papain possesses appreciable nucleophilic character. It is not a necessary requirement that the probe reagent should engage the imidazolium ion of histidine-159 in hydrogen-bonding for the sulphur atom of the interactive system to display nucleophilic character. The single proton-binding site of propyl-S-S-2-Py cannot simultaneously interrupt the active-centre ion pair and provide for rate enhancement as the pH is lowered towards 4. The possible implication of this for the mechanism of papain-catalysed hydrolysis is discussed. 3. The suspected difference in the active centres of papain and ficin (EC 3.4.22.3), which could be a lack in ficin of a carboxy group conformationally equivalent to that of aspartic acid-158 of papain is confirmed. The reactivity of the papain thiol group towards both probe reagents is controlled by two ionizations with pKa close to 4 that are positively co-operative. 4. In the reaction of papain with 2-Py-S-S-2-Py. the reactivity appears to be controlled also by an addition ionization with pKa approx. 5. Possible origins of this additional ionization are discussed. K. The spectral and ionization characteristics of propyl-S-S-2-Py are reported. 6. The reagent reacts rapidly with thiol groups at the sulphur atom distal from the pyridyl ring to provide, at pH values below 9, stoicheiometric release of 2-thiopyridone. This property, together with the ability of the reagent markedly to increase its electrophilicity consequent on protonation, suggests alkyl-2-pyridyl disulphides in general as valuable two-protonic-state reactivity probes with exceptional specificity for thiol groups.

scholarly journals The Role of H2C2O4 and Na2CO3 as Precipitating Agents on The Physichochemical Properties and Photocatalytic Activity of Bismuth Oxide

2020 ◽  
Vol 18 (1) ◽  
pp. 129-137
Author(s):  
Yayuk Astuti ◽  
Rizka Andianingrum ◽  
Abdul Haris ◽  
Adi Darmawan ◽  
Keyword(s):  
Photocatalytic Activity ◽  
Methyl Orange ◽  
Physicochemical Properties ◽  
Bismuth Oxide ◽  
Rate Constants ◽  
Degradation Rate ◽  
Precipitation Method ◽  
Methyl Orange Degradation ◽  
Methyl Orange Dye

AbstractSynthesis of bismuth oxide synthesis through the precipitation method using H2C2O4 and Na2CO3 precipitating agents, identification of physicochemical properties and its photocatalysis activity for methyl orange degradation were conducted. The bismuth oxide synthesis was undertaken by dissolving Bi(NO3)3.5H2O in HNO3, then added precipitating agents to form precipitate. The results showed that bismuth oxide produced by H2C2O4 precipitating agent was a yellow powder containing a mixture of α-Bi2O3 (monoclinic) and β-Bi2O3 (tetragonal), porous with size of 28-85 μm. Meanwhile, the use of Na2CO3 as precipitating agent resulted in bismuth oxide consisting of α-Bi2O3 and β-Bi2O3 and Bi2O4, irregular shape without pore being 40-115 μm in size. Bismuth oxide synthesized with H2C2O4 precipitating agent showed higher photocatalytic activity compared to bismuth oxide synthesized using Na2CO3 on degrading methyl orange dye with degradation rate constants of 2.35x10-5 s-1 for H2C2O4 and 1.81x10-5 s-1 for Na2CO3.

scholarly journals Half-sites oxidation of bovine liver uridine diphosphate glucose dehydrogenase

1978 ◽  
Vol 173 (2) ◽  
pp. 701-704 ◽  
Author(s):  
J S Franzen ◽  
P Marchetti ◽  
R Ishman ◽  
J Ashcom
Keyword(s):  
Catalytic Activity ◽  
Glucose Dehydrogenase ◽  
Bovine Liver ◽  
Thiol Group ◽  
Catalytic Site ◽  
Uridine Diphosphate ◽  
Thiol Groups ◽  
Irreversible Denaturation ◽  
Diphosphate Glucose ◽  
Uridine Diphosphate Glucose Dehydrogenase

6,6-Dithiodinicotinate shows half-of-the-sites reactivity towards the six catalytic-site thiol groups of bovine liver UDP-glucose dehydrogenase. The reagent introduces three intrasubunit disulphide linkages between catalytic-site thiol groups and non-catalytic-site thiol groups and abrogates 60% of the catalytic activity of the hexameric enzyme; excess 2-mercaptoethanol rapidly restores full catalytic activity. These results show the half-of-the-sites behaviour of the enzyme with the reagent and the presence of a non-catalytic-site thiol group capable of forming a disulphide linkage with a catalytic-site thiol group on the same subunit without irreversible denaturation.

scholarly journals A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2'-dipyridyl disulphide as a thiol titrant and reactivity probe

1976 ◽  
Vol 159 (2) ◽  
pp. 245-257 ◽  
Author(s):  
R Norris ◽  
K Brocklehurst
Keyword(s):  
Convenient Method ◽  
Specific Activity ◽  
Class Ii ◽  
High Reactivity ◽  
Class I ◽  
Thiol Groups ◽  
Class Iii ◽  
Active Centre ◽  
Class Ib ◽  
Covalent Chromatography

1. A convenient method of preparation of jack-bean urease (EC3.5.1.5) involving covalent chromatography by thiol-disulphide interchange is described. 2. Urease thus prepared has specific activity comparable with the highest value yet reported (44.5 ± 1.47 kat/kg, Km = 3.32 ± 0.05 mM; kcat. = 2.15 × 104 ± 0.05 × 104s-1 at pH7.0 and 38°C). 3. Titration of the urease thiol groups with 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) and application of the method of Tsou Chen-Lu [(1962) Sci. Sin.11, 1535-1558] suggests that the urease molecule (assumed to have mol.wt. 483000 and ε280 = 2.84 × 105 litre·mol-1-cm-1) contains 24 inessential thiol groups of relatively high reactivity (class-I), six ‘essential’ thiol groups of low reactivity (class-II) and 54 buried thiol groups (class-III) which are exposed in 6M-guanidinium chloride. 4. The reaction of the class-I thiol groups with 2-Py-S-S-2-Py was studied in the pH range 6-11 at 25°C(I = 0.1 mol/l) by stopped-flow spectrophotometry, and the analogous reaction of the class-II thiol groups by conventional spectrophotometry. 5. The class-I thiol groups consist of at least two sub-classes whose reactions with 2-Py-S-S-2-Py are characterized by (a) pKa = 9.1, k = 1.56 × 104M-1·s-1 and (b) pKa = 8.1, k = 8.05 × 102M-1·s-1 respectively. The reaction of the class-II thiol groups is characterized by pKa = 9.15 and k = 1.60 × 102M-1·s-1. 6. At pH values 7-8 the class-I thiol groups consist of approx. 50% class-Ia groups and 50% class-Ib groups. The ratio class Ia/class Ib decreases as the pH is raised according to a pKa value ≥ approx. 9.5, and at high pH the class-I thiol groups consist of at most 25% class-Ia groups and at least 75% class-Ib groups. 7. The reactivity of the class-II thiol groups towards 2-Py-S-S-2-Py is insensitive to the nature of the group used to block the class-I thiols. 8. All the ‘essential’ thiol groups in urease appear to be eeactive only as uncomplicated thiolate ions. The implications of this for the active-centre chemistry of urease relative to that of the thiol proteinases are discussed.

Solvent effect on simple and high yield synthesis of polydichlorophosphazene precursor Cl3PNSiMe3

2021 ◽  
Author(s):  
Elif Büşra Çelebi ◽  
Ferda Hacıvelioğlu
Keyword(s):  
Solvent Effect ◽  
Room Temperature ◽  
Solvent Polarity ◽  
High Yield ◽  
Effect Of Solvent ◽  
Nonpolar Solvents

The effect of solvent polarity on the reaction of PCl5 with (Me3Si)2NH has been investigated and it is found that nonpolar solvents promote the formation of Cl3PNSiMe3, whereas the more polar chloroform gives unusual phosphazenes at room temperature.

Effect of Solvent on Protonation Equilibria of L-Serine and L-Tryptophan in Ethylene Glycol-Water Mixtures

2021 ◽  
Vol 25 (7) ◽  
pp. 124-129
Author(s):  
Ch. Sudhakar ◽  
Allabakshu Shaik ◽  
M. Ramanaiah ◽  
Ch. Nageswara Rao
Keyword(s):  
Dielectric Constant ◽  
Ethylene Glycol ◽  
Protonation Constants ◽  
Statistical Parameters ◽  
Protonation Equilibria ◽  
Effect Of Solvent ◽  
R Factor ◽  
Chemical Models ◽  
Best Fit ◽  
Skewness And Kurtosis

Protonation equilibria of L-serine and L-tryptophan in varying compositions (0.0-50.0 % v/v) of ethylene glycol-water mixtures were investigated pH-metrically. Titrations were performed at 303.0 K and the ionic strength of the medium was maintained at 0.16 mol L-1 using sodium chloride. The protonation constants have been calculated with the computer program MINIQUAD 75 and are selected based on statistical parameters. The best fit chemical models of the protonation equilibria were based on crystallographic R-factor, χ2, skewness and kurtosis. The protonation constants of L-serine and L-tryptophan change linearly with increasing ethylene glycol content. This is attributed to the dielectric constant of the medium.

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