scholarly journals The behaviour of trypsin towards α-N-methyl-α-N-toluene-p-sulphonyl-l-lysine β-naphthyl ester. A new method for determining the absolute molarity of solutions of trypsin

1968 ◽  
Vol 107 (1) ◽  
pp. 97-102 ◽  
Author(s):  
D. T. Elmore ◽  
J. J. Smyth
Keyword(s):  
Methyl Ester ◽  
Convenient Method ◽  
Substrate Concentration ◽  
Competitive Inhibitor ◽  
New Method ◽  
Spectrophotometric Measurement ◽  
Bovine Thrombin ◽  
The Absolute ◽  
Hydrolysis Of

1. α-N-Methyl-α-N-toluene-p-sulphonyl-l-lysine β-naphthyl ester (MTLNE) was synthesized as its hydrobromide and shown to be slowly hydrolysed by bovine pancreatic trypsin. The acylation step, however, is so much faster than deacylation of the acyl-enzyme that spectrophotometric measurement of the ‘burst’ of β-naphthol provides a convenient method for determining the absolute molarity of trypsin solutions. 2. By using the same stock solution of trypsin, application of this method at pH4·0 and pH7·0 as well as that of Bender et al. (1966) at pH3·7 gave concordant results. 3. Provided that [S]0>[E]0, the size of the ‘burst’ is independent of substrate concentration. 4. In the trypsin-catalysed hydrolysis of α-N-toluene-p-sulphonyl-l-arginine methyl ester, MTLNE functions as a powerful non-competitive inhibitor. 5. There is no detectable reaction between MTLNE and either bovine pancreatic α-chymotrypsin at pH4·0 or bovine thrombin at pH6·0.

scholarly journals A new method for determining the absolute molarity of solutions of trypsin and chymotrypsin by using p-nitrophenyl N2-acetyl-N1-benzylcarbazate

1968 ◽  
Vol 107 (1) ◽  
pp. 103-107 ◽  
Author(s):  
D. T. Elmore ◽  
J. J. Smyth
Keyword(s):  
Trypsin Inhibitor ◽  
Soya Bean ◽  
New Method ◽  
Spectrophotometric Measurement ◽  
Bovine Thrombin ◽  
Chymotrypsin Inhibitor ◽  
Tryptophan Residues ◽  
Bean Trypsin Inhibitor ◽  
The Absolute ◽  
Active Centres

1. p-Nitrophenyl N2-acetyl-N1-benzylcarbazate (NPABC) was synthesized and shown to acylate α-chymotrypsin stoicheiometrically; reaction at 25° occurs almost instantaneously at pH7·04 and within 2min. at pH5·04 and there is no observable turnover during 10min. 2. The absolute molarity of solutions of α-chymotrypsin can be determined by spectrophotometric measurement of the p-nitrophenol liberated during the acylation step; the results obtained at pH5·04 and pH7·04 agree with one another and with those determined by the method of Erlanger & Edel (1964). 3. Trypsin reacts stoicheiometrically, but more slowly than α-chymotrypsin, with NPABC, and it, like chymotrypsin, can be spectrophotometrically titrated at pH7·04. At pH5·04, however, reaction between trypsin and NPABC is sufficiently slow for the reagent to be nearly specific for α-chymotrypsin. Specificity for one or other enzyme can be ensured by using soya-bean trypsin inhibitor or the chymotrypsin inhibitor l-1-chloro-3-toluene-p-sulphonamido-4-phenylbutan-2-one. Bovine thrombin does not react with NPABC. 4. Evidence is presented that indicates that acylation of α-chymotrypsin and trypsin by NPABC occurs at the active centres of the enzymes. 5. Evidence was obtained that indicates that one or more tryptophan residues move into a more hydrophobic environment when α-chymotrypsin and trypsin are acylated by NPABC.

scholarly journals Substrate Inhibition in the Hydrolysis of Hippuric Acid Esters by Carboxypeptidase A

1975 ◽  
Vol 53 (2) ◽  
pp. 283-294 ◽  
Author(s):  
Joe Murphy ◽  
John W. Bunting
Keyword(s):  
Substrate Concentration ◽  
Hippuric Acid ◽  
Substrate Inhibition ◽  
Competitive Inhibitor ◽  
Side Chain ◽  
Carboxypeptidase A ◽  
Substrate Activation ◽  
Hydrolysis Of ◽  
Substrate Concentrations ◽  
Acid Esters

The dependence of initial velocity upon substrate concentration has been examined in the carboxypeptidase A catalyzed hydrolysis of the following hippuric acid esters (at pH 7.5, 25°, ionic strength O.5): C6H5CONHCH2CO2CHRCO2H: R=CH3; CH2CH3;(CH2)2CH3; (CH2)3CH3; (CH2)5CH3; CH(CH3)2; CH2CH(CH3)2; C6H5; CH2C6H5. All of these esters display marked substrate inhibition of their enzymic hydrolyses. With the exception of R=CH3, the velocity-substrate concentration profiles for each of these esters can be rationalized by the formation of an E.S2 complex which, independent of the alcohol moiety of the ester, reacts approximately 25 times more slowly than the E.S complex. For most of these esters, the formation of E.S2 approximates ordered binding of the substrate molecules at the catalytic and inhibitory sites. While binding at the catalytic site is markedly dependent on the nature of the R group, binding of a second substrate molecule to E.S is not significantly affected by the nature of the R side chain. For R=C6H5, the D ester is neither a substrate nor a competitive inhibitor of the hydrolysis of the L-ester but can replace the L-ester at the binding site which is responsible for substrate inhibition. The kinetic analysis suggests that this behavior of D and L -enantiomers is also typical of the other esters examined (except possibly R=CH3). For R=CH3 only, substrate activation also seems to occur prior to the onset of substrate inhibition at higher substrate concentrations.

THE HYDROLYSIS OF PHENYLALANINE METHYL ESTER BY BOVINE THROMBIN

1966 ◽  
Vol 44 (7) ◽  
pp. 1051-1059 ◽  
Author(s):  
Edmond R. Cole ◽  
J. L. Koppel ◽  
John H. Olwin
Keyword(s):  
Methyl Ester ◽  
Sodium Citrate ◽  
Deae Cellulose ◽  
Amino Acid Esters ◽  
Bovine Thrombin ◽  
Wide Range ◽  
Autoprothrombin C ◽  
Phenylalanine Methyl Ester ◽  
Hydrolysis Of ◽  
Acid Esters

Thrombin, usually regarded as a trypsin-like enzyme capable of hydrolyzing only esters of the amino acids arginine and lysine, was found to hydrolyze certain other amino acid esters which are considered specific chymotrypsin substrates. L-Phenylalanine methyl ester inhibited the activation of purified bovine prothrombin by autoprothrombin C, Ac-globulin, phospholipid, and calcium. It was subsequently shown that thrombin is capable of hydrolyzing L-phenylalanine methyl ester or L-tyrosine ethyl ester. This activity developed simultaneously with fibrogen clotting activity during activation of purified bovine prothrombin in 25% sodium citrate solutions. Moreover, the activity was closely associated with clotting activity on subsequent chromatography on DEAE-cellulose and Amberlite IRC-50 resin columns. All preparations of bovine thrombin, representing a wide range of purity, which have been examined, exhibited this hydrolyzing activity toward L-phenylalanine methyl ester. Further evidence linking this esterase activity with fibrinogen clotting activity was obtained when both activities were inhibited by 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate, an inhibitor of chymotrypsin.

Hydrolysis of L-Histidine Methyl Ester

1968 ◽  
Vol 19 (03/04) ◽  
pp. 321-333
Author(s):  
E. R Cole
Keyword(s):  
Methyl Ester ◽  
Esterase Activity ◽  
Sodium Citrate ◽  
Deae Cellulose ◽  
Sodium Cholate ◽  
Michaelis Constant ◽  
Bovine Thrombin ◽  
Optimum Ph ◽  
Autoprothrombin C ◽  
Hydrolysis Of

SummaryThe hydrolysis of L-histidine methyl ester (HME) by bovine thrombin preparations has been investigated. Activation of purified bovine prothrombin in 25% sodium citrate solution resulted in the simultaneous development of fibrinogen clotting activity and of TAME and HME esterase activities. Most of the HME esterase activity was identified with fibrinogen clotting activity on sequential chromatography of activated prothrombin on DEAE-cellulose and Amberlite CG-50 resin columns, although some HME esterase activity could be demonstrated in concentrates of the autoprothrombin C fraction. The optimum pH for HME hydrolysis by thrombin was found at 7.6 in phosphate and Tris buffered reactions. Tris buffer and other amines depress HME esterase activity of thrombin, while sodium cholate accelerates the reaction. The Michaelis constant, Km, was estimated to be 0.134 M at pH 7.6 in phosphate buffer and at 37° C.

The Effects of Biguanides on Thrombokinase, Thrombin and Trypsin

1975 ◽  
Vol 34 (01) ◽  
pp. 246-255
Author(s):  
Phyllis S Roberts ◽  
Patricia B Fleming
Keyword(s):  
Methyl Ester ◽  
Enzymatic Activity ◽  
Test Period ◽  
Factor X ◽  
Bovine Thrombin ◽  
Slow Reaction ◽  
Bovine Trypsin ◽  
Rate Of Hydrolysis ◽  
Almost All ◽  
Hydrolysis Of

SummaryA purified preparation of bovine thrombokinase (activated Factor X) loses the ability to hydrolyze TAME (p-toluenesulfonyl-L-arginine methyl ester) when it is incubated at 37° in 0.25 M Tris. HCl buffer, pH 7.4 with lauroxypropyl biguanide, N1, N5-dimethyl, N1-lauroxypropyl biguanide, N1-p-chlorophenethyl, N5-phenethyl biguanide, or N1-methyl, N1-p-chlorobenzyl, N5-o,p-dichlorobenzyl biguanide. Activity is lost much more slowly when 0.15 M NaCl is also present. Lauroxypropyl biguanide is the most potent of the compounds tested, 0.22 mM causing thrombokinase to lose almost all of its activity in about 30 minutes at 37° in pH 7.4 buffered saline.Topical bovine thrombin also loses activity when incubated with either of the lauroxypropyl biguanides but not with the diphenethyl or the dibenzyl compound. Instead, the latter biguanides accelerate thrombin’s hydrolysis of TAME. The percent acceleration is not affected or only slightly decreased by the presence of 0.15 M NaCl or KCl, and it is also unaffected by incubating the enzyme with the compounds in buffered saline for 4 to 120 minutes.Purified bovine trypsin is stabilized by both lauroxypropyl and the diphenethyl biguanide when incubated at 37° in pH 7.4 buffered saline for the 60 minute test period but neither compound has any effect on its rate of hydrolysis of TAME.It is postulated that the enzymes first react rapidly and reversibly with all of the test biguanides and, depending upon the enzyme and the substrate, the rate of hydrolysis of the substrate is unaffected, accelerated or inhibited. The lauroxypropyl biguanides also undergo a second, slower reaction with both thrombokinase and thrombin that produces loss of enzymatic activity. The dibenzyl and diphenethyl biguanides also undergo this second slow reaction with thrombokinase but not with thrombin, and none of the biguanides undergo this second reaction with trypsin.

The Effects of Amines on the Esterase Activities of Thrombin and Thrombokinase and on Several Clotting Tests

1974 ◽  
Vol 31 (02) ◽  
pp. 309-318
Author(s):  
Phyllis S Roberts ◽  
Raphael M Ottenbrite ◽  
Patricia B Fleming ◽  
James Wigand
Keyword(s):  
Choline Chloride ◽  
Polymerization Process ◽  
Ammonium Bromide ◽  
Bovine Thrombin ◽  
One Stage ◽  
Human Proteins ◽  
Rate Of Hydrolysis ◽  
The One ◽  
Hydrolysis Of Esters ◽  
Hydrolysis Of

Summary1. Choline chloride, 0.1 M (in 0.25 M Tris. HCl buffer, pH 7.4 or 8.0, 37°), doubles the rate of hydrolysis of TAME by bovine thrombokinase but has no effect on the hydrolysis of this ester by either human or bovine thrombin. Only when 1.0 M or more choline chloride is present is the hydrolysis of BAME by thrombokinase or thrombin weakly inhibited. Evidence is presented that shows that these effects are due to the quaternary amine group.2. Tetramethyl ammonium bromide or chloride has about the same effects on the hydrolysis of esters by these enzymes as does choline chloride but tetra-ethyl, -n.propyl and -n.butyl ammonium bromides (0.1 M) are stronger accelerators of the thrombokinase-TAME reaction and they also accelerate, but to a lesser degree, the thrombin-TAME reaction. In addition, they inhibit the hydrolysis of BAME by both enzymes. Their effects on these reactions, however, do not follow any regular order. The tetraethyl compound is the strongest accelerator of the thrombokinase-TAME reaction but the tetra-ethyl and -butyl compounds are the strongest accelerators of the thrombin-TAME reaction. The ethyl and propyl compounds are the best (although weak) inhibitors of the thrombokinase-BAME and the propyl compound of the thrombin-BAME reactions.3. Tetra-methyl, -ethyl, -n.propyl and -n.butyl ammonium bromides (0.01 M) inhibit the clotting of fibrinogen by thrombin (bovine and human proteins) at pH 7.4, imidazole or pH 6.1, phosphate buffers and they also inhibit, but to a lesser degree, a modified one-stage prothrombin test. In all cases the inhibition increases regularly as the size of the alkyl group increases from methyl to butyl. Only the ethyl com pound (0.025 M but not 0.01 M), however, significantly inhibits the polymerization of bovine fibrin monomers. It was concluded that inhibition of the fibrinogen-thrombin and the one-stage tests by the quaternary amines is not due to any effect of the com pounds on the polymerization process but probably due to inhibition of thrombin’s action on fibrinogen by the quaternary amines.

What Women Say They Want versus What Men Imagine They Do: A new and convenient method reveals novel insights about self-reported vs. perceived preferences

2019 ◽  
Author(s):  
Carl Pierre Jago ◽  
Karen R. Dobkins
Keyword(s):  
Convenient Method ◽  
Physical Attractiveness ◽  
Romantic Partner ◽  
Self Report ◽  
New Method ◽  
Wealth Status ◽  
Men And Women ◽  
Potential Partner ◽  
Self Reports ◽  
Opposite Gender

To appeal to the opposite gender, previous research indicates that men emphasize their wealth, status, and ambition, whereas women emphasize their physical attractiveness. Such behavior seems surprising given previous surveys in which men and women reported these traits to be less important than others such as trustworthiness, intelligence, and warmth. We addressed one potential reason for any disconnect, which is that men’s and women’s beliefs about what the opposite gender prefers are misguided—according to the opposite genders’ self-reports. Using a new method, we asked participants to both self-report the traits they prefer in a romantic partner and to indicate what they imagine the opposite gender prefers. The results reveal striking discrepancies between what people report wanting in a potential partner and what the opposite gender imagines they want. Additionally, women appear to be better at imagining men’s preferences, and we discuss several reasons why this might be the case.

Variation in Quantity of Methanol Recovered from Raisins

1963 ◽  
Vol 46 (2) ◽  
pp. 341-343
Author(s):  
M Alice Brown ◽  
James R Woodward ◽  
Floyd DeEds
Keyword(s):  
Methyl Ester ◽  
Esterase Activity ◽  
Enzymic Hydrolysis ◽  
Methanol Content ◽  
Naturally Occurring ◽  
Pectin Esterase ◽  
Hydrolysis Of

Abstract The amount of naturally occurring methanol in fruit must be known so that the quantity left as fumigation residue can be determined. In a study of methanol content of raisins, which had given inconsistent results, the raisins were subjected to different conditions of treatment immediately prior to methanol determination. Conditions that favored pectin esterase activity gave higher values for methanol content than conditions known to inactivate enzymes. Evidence was also obtained that both chemical and enzymic hydrolysis of methyl ester groups of pectic materials occur during analysis.

Preparation and structural analysis of (±)-threo-ritalinic acid

2013 ◽  
Vol 69 (11) ◽  
pp. 1225-1228 ◽  
Author(s):  
Sara Wyss ◽  
Irmgard A. Werner ◽  
W. Bernd Schweizer ◽  
Simon M. Ametamey ◽  
Selena Milicevic Sephton
Keyword(s):  
Methyl Ester ◽  
Free Acid ◽  
Nmr Analysis ◽  
Intermolecular Hydrogen Bonds ◽  
X Ray ◽  
X Ray Crystallography ◽  
Ph Values ◽  
Ritalinic Acid ◽  
Methyl Phenidate ◽  
Hydrolysis Of

Hydrolysis of the methyl ester (±)-threo-methyl phenidate afforded the free acid in 40% yield,viz.(±)-threo-ritalinic acid, C13H17NO2. Hydrolysis and subsequent crystallization were accomplished at pH values between 5 and 7 to yield colourless prisms which were analysed by X-ray crystallography. Crystals of (±)-threo-ritalinic acid belong to theP21/nspace group and form intermolecular hydrogen bonds. An antiperiplanar disposition of the H atoms of the (HOOC—)CH—CHpygroup (py is pyridine) was found in both the solid (diffraction analysis) and solution state (NMR analysis). It was also determined that (±)-threo-ritalinic acid conforms to the minimization of negativegauche+–gauche−interactions.

Sign in / Sign up

Export Citation Format

Share Document