Method for determining nitrogen-containing cationic surface-active agents using butyrylcholinesterase

The biochemical method for determination of cetyltrimethyl ammonium or cetylpyridinium, both being nitrogenated cationic surfactants, has been devised by using horse blood serum butyrylcholinesterase as analytical reagent. The method streams from the fact that surfactants tested are inhibitors of butyrylcholinesterase hydrolysis of butyrylcholin, a cationic substrate, but in this case they activate enzymatic hydrolysis of 1-naphthylacetate, a neutral substrate. Presence two opposite effects enlarges reliability to identifications. Use the sensitive fluorimetric method to registrations of activation of hydrolysis a substrate 1-naphtylacetate vastly to reduce the threshold of determination of surfactants above.     

The effects of ammonium sulfate and acid on horse and human serum butyrylcholinesterase (EC 3.1.1.8)

1. Results of laboratory experiments which compared horse and human serum butyrylcholinesterase (EC 3.1.1.8) with respect to their acid inactivation and ammonium sulfate protection show: 2. Horse serum butyrylcholinesterase is more resistant to inactivation at pH 3.0 than human serum butyrylcholinesterase. 3. The loss of activity at pH 3.0 for both horse and human butyrylcholinesterase does not follow first order kinetics. 4. Both human and horse serum butyrylcholinesterase are protected from pH 3.0 inactivation by ammonium sulfate concentrations up to 33% saturation (1.37 M).     

A subunit-sized butyrylcholinesterase present in high concentrations in pooled rabbit serum

A butyrylcholinesterase of mol.wt. approx. 83000 was observed in pooled rabbit serum. The enzyme was named monomeric butyrylcholinesterase to distinguish it from the larger oligomeric butyrylcholinesterase of horse and human serum whose subunits are the same size as the monomeric enzyme. The active-site concentration of monomeric butyrylcholinesterase in the pooled serum was 0.18mum, which is five times the concentration of butyrylcholinesterase in pooled horse serum. This was surprising, since the horse serum is regarded as a rich source of butyrylcholinesterase, whereas rabbit serum is not generally thought to contain significant amounts of any butyrylcholinesterase. The explanation, in large part, was the relatively low k(cat.) of the monomeric enzyme, which was approx. 57s(-1) with butyrylthiocholine as substrate and is one-thirtieth of the comparable k(cat.) of horse butyrylcholinesterase. The substrate specificity of monomeric butyrylcholinesterase also differed significantly from that of horse and human butyrylcholinesterase. For example, with the monomeric enzyme, the hydrolysis of 1mm-acetylthiocholine was only 4% the rate for 1mm-butyrylthiocholine, whereas human and horse butyrylcholinesterases hydrolysed 1mm-acetylthiocholine at 50% of the rate for 1mm-butyrylthiocholine. Moreover, monomeric butyrylcholinesterase generally hydrolysed aromatic esters more rapidly than choline esters, whereas the reverse is true of the butyrylcholinesterases. To facilitate the study of monomeric butyrylcholinesterase, it was separated from the larger butyrylcholinesterase and acetylcholinesterase, also present in rabbit serum, and purified 89-fold by fractionation with (NH(4))(2)SO(4) and ion-exchange chromatography.     

Inhibition of Catalytic Activity of Horse Blood Serum Butyrylcholinesterase by High Concentrations of a Substrate,N-methyl-N-(β-acetoxyethyl)piperidinium

The kinetics of hydrolysis of N-methyl-N-(β-acetoxyethyl)-piperidinium by highly purified horse blood serum butyrylcholinesterase is studied at pH 7.5 and 25°C. A pronounced inhibition of catalytic activity of this enzyme by high concentrations of the substrate is revealed. The substrate inhibition can be explained either by interaction of the acylated enzyme with substrate with formation of the corresponding inactive complex ES'S or by sorption of substrate outside the enzyme active center and the resulted conformational changes of the enzyme molecule.     

Effect of cetyltrimethylammonium on the human blood cholinesterases activity

The influence of cationic detergent cetyltrimethylammonium on the human blood cholinesterases activity (erythrocyte acetylcholinesterase and plasma butyrylcholinesterase) in reactions of hydrolysis of alpha-thionaphthylacetat and acetylthiocholine is studied. It is shown, that cetyltrimethylammonium is reversible effector for both cholinesterases. This compound competitively inhibited enzymatic hydrolysis of acetylthiocholine by both cholinesterases, and in the reactions of enzymatic hydrolysis alpha-thionaphthylacetat display as the synergistic activator--in experiments with butyrylcholinesterase, and as the reversible inhibitor--in experiments with acetylcholinesterase. Kinetic constants in reaction of acetylcholinesterase inhibition by cetyltrimethylammonium defined by means of different substrates--alpha-thionaphthylacetat and acetylthiocholin. They are close among themselves and amount (2.5 +/- 0.3) x 10(-5) and (2.8 +/- 0.3) x 10(-5) M, accordingly. Butyrylcholinesterase was more sensitive to influence of cetyltrimethylammonium. The kinetic constants defined for this enzyme by the effect of inhibition of acetylthiocholin hydrolysis or activation of alpha-thionaphthylatcetat hydrolysis, are also close among themselves and amount (3.9 +/- 0.4) x 10(-6) and (4.4 +/- 0.4) x 10(-6) M, accordingly.     

Kinetics of 13 new cholinesterase inhibitors

Kinetics of hydrolysis of acetylcholine and acetylthiocholine by two types of acetylcholinesterase and butyrylcholinesterase inhibited by 13 new inhibitors (5 carbamates and 8 carbazates--hydrazinium derivatives) was measured in vitro in a batch reactor at 25 degrees C, pH 8, ionic strength 0.11 M and enzyme activity 3.5 U by four nondependent analytical methods. Sevin, rivastigmin (Exelon) and galantamin (Reminyl) served as comparative inhibiting standards. Kinetics of hydrolyses inhibited by all studied carbamates, sevin, carbazates (with exceptions) and rivastigmin (with exceptions) can be simulated by the competitive inhibition model with irreversible reaction between enzyme and inhibitor. Galantamin does not fulfil this model. In positive simulations, the value of inhibition (carbamoylation) rate constant k3 was calculated, describing the reaction velocity between the given enzyme and inhibitor. Physiologically important hydrolyses of acetylcholine catalyzed by acetylcholinesterase from electric eel or bovine erythrocytes and butyrylcholinesterase from horse plasma can be most quickly inhibited by carbamoylation of the mentioned enzymes by the 3-N,N-diethylaminophenyl-N'-(1-alkyl) carbamates 4 and 5. Probably this is due to a long enough hydrocarbon aliphatic substituent (hexyl and octyl) on the amidic nitrogen atom. The tested carbazates failed as inhibitors of cholinesterases. The regeneration ability of the inhibited enzymes was not measured.     

Study of hydrolysis of aminoalcohol ethers, phenol and choline under the action of horse blood serum cholinesterase]

Hydrolysis of ethers of saturated and unsaturated alcohols and ethers, e.g. phenol and choline, under the action of horse blood serum cholinesterase, was studied. The reactivity towards enzymatic hydrolysis is decreased due to a greater length of the chain in the alcohol residue of the benzoic acid aminoethers; at nCH2 = 4 the compound is a poor substrate. An increase in nydrophobicity of the acyl residue of the ether molecule also leads to a decrease in the Vmax and Km values. In case of cholinesterase substrates, an increase in the molecule hydrophobicity results in an increase of its non-productive absorption on the active surface of the enzyme, which decreases its hydrolysis. Aminobutynol benzoates are hydrolyzed by cholinesterase more rapidly as compared to the ethers of corresponding aminobutanols and their homologs.     

Flow microcalorimetric study of butyrylcholinesterase kinetics and inhibition

The enzymatic hydrolysis of butyrylcholine, catalyzed by horse serum butyrylcholinesterase (EC 3.1.1.8), was studied at 37 degrees C in Tris buffer (pH 7.5) by flow microcalorimetry. A convolution procedure, using the Gamma distribution to represent the impulse response of the calorimeter, was developed to analyze the microcalorimetric curves. After correction for buffer protonation, the hydrolysis reaction was found to be slightly endothermic, with Delta H=+9.8 kJ mol(-1). Enzyme kinetics was studied with both the differential and integrated forms of the Michaelis equation with equivalent results: Michaelis constant K(m)=3.3mM, catalytic constant k(cat)=1.7 x 10(3)s(-1), bimolecular rate constant k(s)=5.1 x 10(5)M(-1)s(-1). The reaction product, choline, was found to be a competitive inhibitor with a dissociation constant K(i)=9.1mM. Betaine had a slightly higher affinity for the enzyme, but the inhibition was only partial. This study confirms the usefulness of microcalorimetry for the kinetic study of enzymes and their inhibitors.     

Cholinesterase hydrolysis of acetylcholine derivatives with different structures of the ammonium grouping]

15 acetoxyethylenammonium compounds are studied as substrates for acetylcholinesterase (ACE) from bovine erythrocytes and for butyrylcholinesterase (BCE) from horse serum. Substitution of methyl groups of the ammonium grouping with other radicals and incorporation of onium nitrogen in the cycle resulted in the decrease of the hydrolysis rate under the action of BCE and ACE, the effect of BCE being more pronounced. The rate of the hydrolysis of N-acetoxyethylene-N-methylpiperidine iodide in the presence of ACE was 65 times as much as in the presence of BCE. This compound is a new specific substrate of ACE. Dipropylmethyl derivative turned not to be a good substrate for both enzymes. Dibutylmethyl and pyridinic derivatives were not attacked by ACE and BCE. Kinetic analysis of the compounds listed is performed, taking account of non-productive sorbtion. Possible role of hydrofobic regions in the orientation of substrates on the active surface of ACE and BCE is discussed.     

How the various substrates activate the process of enzymatic hydrolysis by different cholinesterases

Kinetic analysis of the activating effect of substrate on the cholinesterase catalysis is performed. There are determined values of coefficient of activation A in the pH zone 5.0-7.5 for the process of hydrolysis of acetylcholine, indophenylacetate (IPA), and 2,6-dichlorophenolindophenylacetate (DIPA) by cholinesterase (ChE) of horse blood serum, as well as of IPA and DIPA by ChE of optical ganglia of the Pacific squid Todarodes pacificus. The phenomenon of activation has not been revealed at hydrolysis of phenylacetate by the horse blood serum ChE. The conclusion is made that the cause of the activating effect of substrate on the process of enzymatic hydrolysis by ChEs of different origin is the presence of the onium grouping in the structure of substrates.     

Inhibition of cholinesterases by quaternary phosphonium compounds

Alkyl tributylphosphonium and triphenylphosphonium derivatives as well as tetraphenylphosphonium were first studied as inhibitors of acetylcholinesterase of human blood erythrocytes and butyrylcholinesterase of horse blood serum. The inhibition is reversible, of mixed type, with a different contribution of competitive and uncompetitive components. The value of the inhibitory effect is essentially dependent on the structure of phosphonium compounds, especially in experiments with butyrylcholinesterase: allyltriphenylphosphonium is 290 times as strong enzyme inhibitor as methyltributylphosphonium. Hexyltributylphosphonium is identical to hexyltributylammonium in both the pattern and efficiency of the inhibitory action on cholinesterases.     

How various substrates activate the process of enzymatic hydrolysis by different cholinesterases

Kinetic analysis of the activating effect of substrate on the cholinesterase catalysis is performed. There are determined values of coefficient of activation A in the pH zone 5 for the process of hydrolysis of acetylcholine, indophenylacetate (IPhA), and 2,6-dichlorophenolindoph enylacetate (DIPhA) by cholinesterase (ChE) of horse blood serum, as well as of IPhA and DIPhA by ChE of optical ganglia of the Pacific squid Todarodes pacificus. The phenomenon of activation has not been revealed at hydrolysis of phenylacetate by the horse blood serum ChE. The conclusion is made that the cause of the activating effect of substrate on the process of enzymatic hydrolysis by ChEs of different origin is the presence of the onium grouping in the structure of substrates.     

Alkylammonium chlorobenzoates are a new group of ester-containing reversible inhibitors of cholinesterases of different animals

Interaction with cholinesterases (ChEs) of nine specially synthesized derivatives of dimethylaminoalkyl esters of 2-chloro-and 2,4-dichlorobenzoic acids and their iodoalkylates is studied. Used as enzyme sources were partially purified preparations of acetylcholinesterase (AChE) from human erythrocytes and butyrylcholinesterase (BChE) from horse blood serum, as well as water homogenates of the frog Rana temporaria brain and of the Pacific squid Todarodes pacificus optical ganglia. The studied benzoates failed to be hydrolyzed by the studied ChEs at the enzyme concentrations exceeding 10 times those used for determination of the acetylthiocholine hydrolysis rate. These compounds have turned out to be reversible inhibitors of ChEs of the mixed-noncompetitive type of action. Effects on the anticholinesterase activity of such structural elements of the inhibitors as the acidic part of the benzoate molecule, length of polymethylene chain in the molecule alcoholic part, and the structure of ammonium group are studied. This study has allowed revealing some peculiarities of the reaction capability of vertebrate and invertebrate ChEs.     

Influence of the substrate nature, ethanol and phosphate buffer on the butyrylcholinesterase hydrolysis retardation by reversible inhibitors]

The reversible inhibition of horse blood serum butyrylcholinesterase (Ce 3.1.1.8) hydrolysis of ion substrates of acetyl- and butyrylthiocholines and non-ion substrate of indophenylacetate by N-methyl-4-piperidinylbenzylate and tacrine (1,2,3,4,-tetrahydro-9-aminoacridine) and phosphate buffer and ethanol influence on this process are investigated. The values of competitive Ki, uncompetitive K'i and generalized K sigma inhibitory constants are determined. It is shown that the inhibition effect and reversible inhibition type depend not only on the inhibitor and substrate nature but also on the phosphate buffer concentration and ethanol presence in the reaction mixture.     

Cholinesterase activity in tissues of some crab species from the Japan Sea

The comparative study of the cholinesterase activity in some crab species was carried out for the first time with use of a set of thiocholine substrates. The substrate specificity was studied in stellar nerve, heart, and hemolymph of three crab species. The crab hemolymph was shown to be characterized by the highest enzyme activity. The enzyme from various crab organs has different structure o substrate specificity. Properties of crab enzymes were compared with acetylcholinesterase (AChE) of human blood erythrocytes, butyrylcholinesterase (BuChE) of horse blood serum, enzyme of squids and bivalve molluscs. The obtained data allow the conclusion to be made on differences in properties of enzymes both at the interspecies and at the tissue levels.     

Substrate activation and thermal denaturation kinetics of the tetrameric and the trypsin-generated monomeric forms of horse serum butyrylcholinesterase

Native horse serum butyrylcholinesterase (acylcholine acylhydrolase; EC 3.1.1.8) is a tetrameric enzyme which can dissociate after a limited proteolysis by trypsin into three additional molecular forms, including the monomeric entity. The trypsin-generated monomer of butyrylcholinesterase, isolated by ultracentrifugation on sucrose gradient, is stable and allows the relations between the polymeric structure of butyrylcholinesterase and its kinetic characteristics to be approached, e.g., substrate activation and complex thermal denaturation curves. The trypsin-generated monomer of butyrylcholinesterase behaves with identical kinetic parameter values as the native tetrameric enzyme. On the other hand, the thermal denaturation of the native tetrameric butyrylcholinesterase does not follow first-order kinetics, but may be described by a sum of exponential terms. This behavior is not due to the polymeric nature of butyrylcholinesterase but seems to be related to a structural heterogeneity induced by the heat treatment.     

Evaluation of organosolv processes for the fractionation and modification of corn stover for bioconversion

Corn stover was pretreated for compositional fractionation and structural modification for maximum conversion of carbohydrate to soluble sugars. The process scheme consisted of three steps: (1) mild prehydrolysis in dilute sulfuric acid, (2) delignification with various organosolv solvents, and (3) enzymatic hydrolysis in an agitated bead reactor. Prehydrolysis of corn stover can be achieved at temperatures ranging from 95 to 120 degrees C, which is a much milder condition than must be applied to wood. Various organosolv solvents, including several alcohols with acid as catalyst, ethylene glycol, and its derivatives, and amines were used for delignification of the prehydrolyzed corn stover. Aromatic alcohols were found to be more effective in solubilizing the prehydrolyzed corn stover than were the aliphatic alcohols. Butanol was the most effective among the aliphatic alcohols; on the other hand, phenol was the best among the aromatic alcohols. Ethylene glycol, methylcellosolv, and ethylcellosolv were effective in solubilizing the prehydrolyzed corn stover but not for enhancing the enzymatic hydrolysis. Various amines achieved delignification at the mild temperature of 95 degrees C, but they tended to solubilize substantial amounts of carbohydrate in addition to lignin. n-Butylamine was effective in enhancing the conversion during enzymatic hydrolysis; it was a good delignifying agent as well as one that achieved a concomitant swelling of the cellulose structure. The low enzymic conversion (20-37%) of prehydrolyzed and solvent-extracted corn stover that was achieved implies that lignin is not the only major barrier for enzymatic hydrolysis. Modification of cellulose structure also should be accomplished to achieve a high degree of conversion. Enzymatic hydrolysis in the agitated bead system increased the rate and extent of conversion of corn stover substantially compared to systems without beads.     

Inhibition and protection of cholinesterases by methanol and ethanol

The cholinesterases have been investigated in terms of the effects of methanol and ethanol on substrate and carbamate turnover, and on their phosphorylation. It was found: 1) that at low substrate concentrations the two alcohols inhibit all three tested cholinesterases and that the optimum activities are shifted towards higher substrate concentrations, but with a weak effect on horse butyrylcholinesterase; 2) that methanol slows down carbamoylation by eserine and does not influence decarbamoylation of vertebrate and insect acetylcholinesterase and 3) that ethanol decreases the rate of phosphorylation of vertebrate acetylcholinesterase by DFP. Our results are in line with the so-called 'approach-and-exit' hypothesis. By hindering the approach of substrate and the exit of products, methanol and ethanol decrease cholinesterase activity at low substrate concentrations and allow for the substrate inhibition only at higher substrate concentrations. Both effects appears to be a consequence of the lower ability of substrate to substitute alcohol rather than water. It also seems that during substrate turnover in the presence of alcohol the transacetylation is negligible.     

Ammonium Compounds with Localized and Delocalized Charge as Reversible Inhibitors of Cholinesterases of Different Origin

Five derivatives of benzimidazole, compounds with delocalized charge in cationic group, are studied and turned out to be reversible inhibitors of hydrolysis of acetylthiocholine under action of acetylcholinesterase from human erythrocytes, butyrylcholinesterase from horse blood serum, and cholinesterases from brain of the brown frog Rana temporaria and from optical ganglion of the Pacific squid Todarodes pacificus. It was only for acetylcholinesterase from erythrocyte as well as (with propyonylthiocholine as substrate) from squid that sensitivity to the studied benzimidazole derivatives correlated with degree of localization of the charge in the cationic group; this confirms the current concepts of functioning of the enzyme active center. A comparative study of 9 ammonium inhibitors with localized cation in their molecules, including the complete sterical analogue of the benzimidazole derivatives, benzimidazolinium iodide, has revealed both quantitative and qualitative differences.     

Ligands of cholinesterases of ephedrine and pseudoephedrine structure

The paper is a review of literature data on interaction of erythrocytic acetylcholinesterase and of mammalian blood serum butyrylcholinesterase with a group of isomeric complex ester derivatives (acetates, propionates, butyrates, valerates, and isobutyrates) of bases and iodomethylates of ephedrine and its enantiomer pseudoephedrine. For 20 alkaloid monoesters, parameters of enzymatic hydrolysis are determined and their certain specificity toward acetylcholinesterase is revealed, whereas 5 diesters of iodomethylates of pseudoephedrine were submitted to hydrolysis only by butyrylcholinesterase. It turned out that 20 alkaloid diesters and 10 trimethylammonium derivatives were uncompetitive reversible inhibitors of acetylcholinesterase and competitive inhibitors of butyrylcholinesterase. The performed for the first time isomer and enantiomer analysis “structure—efficiency” has shown that in most caes it is possible to state the greater complementarity of catalytical surface of enzymes for ligands of pseudoephedrine structure, such differentiation being more often manifested.