PHOTOREGULATION OF α-CHYMOTRYPSIN ACTIVITY BY ISOMERIZABLE INHIBITORS. STUDY OF THE ACTION MECHANISM*

Abstract— The N- p -phenylazophenyl-N-phenylcarbamyl chloride (PAPC) in its cis form is five times more active as inhibitor of α-chymotrypsin than in the trans form. In the present work, derivatives of PAPC have been synthesized. Each of these new compounds is photoisomerizable and is an inhibitor (in the cis and in the trans form) of α-chymotrypsin. The cis isomer is always more active than the trans. The m -methyl derivative is 17.5 times more active in the cis form than in the trans , whereas, for the para -substituted compound, this ratio is only 3.5.
Several hypotheses can explain this difference of activity between the cis and trans isomers: (1) steric hindrance towards the trans isomer, (2) lower affinity of the enzyme for the trans isomer, (3) higher reactivity of the complex formed between the enzyme and the cis form of the inhibitor. These hypotheses are discussed.     

Immobilization of α-chymotrypsin into the poly(N,N-diallyl-N,N-didodecyl ammonium bromiae)/surfactant nanocapsules

The biocatalytic systems from nanocapsules containing α-chymotrypsin in the inner aqueous cavities have been prepared. They can act in both the organic solvent and the aqueous medium. For such encapsulation, the reversed hydrated micelles from N,N-diallyl-N,N-didodecyl ammonium bromide (DDAB) in cyclohexane (w₀ = 22), including α-chymotrypsin, have been polymerized by UV initiation. After precipitation by acetone, these nanocapsules were moved into the aqueous medium with the aid of ionic, AOT, or nonionic, Brij-97, surfactants. In this case, the unilamellar liposomes were formed. They have the inner monolayer from the poly-DDAB network, and the outer one predominantly from surfactant molecules. According to the light-scattering data, the average outer diameter of nanocapsules equals to 20 nm. The vesicular “coated” α-chymotrypsin was used for study of enzymatic activity. It has been shown using the integral form of the Michaelis-Menten equation, that by encapsulation of α-chymotrypsin the value of the Michaelis constant, K_m, increases by a factor of 1.8 by the ATEE hydrolysis. However, the value of the maximal velocity, V_max, decreases by a factor of 1.7. Encapsulated α-chymotrypsin has a high thermostability keeping its own activity up to 80°C. The polymer network blocks the conformational transitions of enzyme molecule by heating of a system.     

LASER PHOTOLYSIS STUDY OF TRIPLET EOSIN AND THIONINE REACTIONS IN PHOTOSENSITIZED OXIDATIONS

Abstract —Nanosecond laser photolysis was used to determine the rate constants of quenching (including chemical reactions) of the triplet states of eosin and thionine, at pH 7, by imidazole, l-histidine, l-methionine, l-phenylalanine, l-tryptophan, 3-indole-acetic acid, cytidine, guanosine, α-chymotrypsin, lysozyme, allylthiourea, thiourea, p -nitrosodimethylaniline, I^- N₃^- and Fe(CN)₆^4-. The quenching rate constants for thionine were found to be up to three orders of magnitude higher than those for eosin. The mechanism of photosensitized oxygenations (free radical vs singlet oxygen) is discussed on the basis of the present results.     

Reverse hydrolysis reaction in aqueous medium without any cosolvent

The synthesis of L-tyrosine fructosyl ester, from fructose and L-tyrosine methyl ester, was carried out by a transesterification reaction catalyzed by α-chymotrypsin in water without cosolvent. The effect of fructose concentration and temperature for the transesterification reaction were determined on both specific activities and product yield. The influence of the presence of fructose has been studied regarding α-chymotrypsin and L-tyrosine fructosyl ester stabilities. It appeared that an increase of temperature enhanced enzyme activity but slumped the product yield because of the very weak stability of tyrosine fructosyl ester.     

Controlled hydrolysis of cheese whey proteins using trypsin and α-chymotrypsin

This study examined the production of protein hydrolysates with controlled composition from cheese whey proteins. Cheese whey was characterized and several hydrolysis experiments were made using whey proteins and purified β-lactoglobulin, assubstrates, and trypsin and α-chymotrypsin, as catalysts, at two tem peratures and several enzyme concentrations. Maximum degrees of hydrolysis obtained experimentally were compared to the theoretical values and peptide compositions were calculated. For trypsin, 100% of yield was achieved; for α-chymotrypsin, hydrolysis seemed to be dependent on the oligopeptide size. The results showed that the two proteases could hydrolyze β-lactoglobulin. Trypsin and α-chymotrypsin were stable at 40°C, but a sharp decrease in the protease activity was observed at 55°C.     

MODIFICATION OF α-CHYMOTRYPSIN USING A WATER-SOLUBLE PHOTO-FENTON REAGENT

Abstract— -Modification of an enzyme, α-chymotrypsin, was examined by using a water-soluble photo-Fenton reagent. By photoirradiation of the enzyme with the reagent, which can occupy a binding site of the enzyme, a tryptophan residue in the vicinity of the active site was oxidized to N -formylkynurenine. Concurrently, the catalytic properties of the enzyme were largely changed: the K _m was increased and the k _catwas decreased. The decrease in k _cat for a specific amide substrate was the most significant among the esters and amides examined. The water-soluble photo-Fenton reagent would be useful to chemically modify relatively limited regions in biomolecules.     

Coordinatively unsaturated semisandwich complexes of ruthenium with phosphinoamine ligands and related species: a complex containing (R,R)-1,2-bis((diisopropylphosphino)amino)cyclohexane in a new coordination form kappa(3)P,P',N-eta(2)-P,N

The syntheses of the chloro complexes [Ru(eta5-C5R5)Cl(L)] (R = H, Me; L = phosphinoamine ligand) (1a-d) have been carried out by reaction of [(eta5-C5H5)RuCl(PPh3)2] or {(eta5-C5Me5)RuCl}4 with the corresponding phosphinoamine (R,R)-1,2-bis((diisopropylphosphino)amino)cyclohexane), R,R-dippach, or 1,2-bis(((diisopropylphosphino)amino)ethane), dippae. The chloride abstraction reactions from these compounds lead to different products depending on the starting chlorocomplex and the reaction conditions. Under argon atmosphere, chloride abstraction from [(eta5-C5Me5)RuCl(R,R-dippach)] with NaBAr'4 yields the compound [(eta5-C5Me5)Ru(kappa3P,P'-(R,R)-dippach)][BAr'4] (2b) which exhibits a three-membered ring Ru-N-P by a new coordination form of this phosphinoamine. However, under the same conditions the reaction starting from [(eta5-C5Me5)RuCl(dippae)] yields the unsaturated 16 electron complex [(eta5-C5Me5)Ru(dippae)][BAr'4] (2d). The bonding modes of R,R-dippach and dippae ligands have been analyzed by DFT calculations. The possibility of tridentate P,N,P-coordination of the phosphinoamide ligand to a fragment [(eta5-C5Me5)Ru]+ is always present, but only the presence of a cyclohexane unit in the ligand framework converts this bonding mode in a more favorable option than the usual P,P-coordination. Dinitrogen [(eta5-C5R5)Ru(N2)(L)][BAr'4] (3a-d) and dioxygen complexes [(eta5-C5H5)Ru(O2)(R,R-dippach)][BPh4] (4a) and [(eta5-C5Me5)Ru(O2)(L)][BPh4] (4b,d) have been prepared by chloride abstraction under dinitrogen or dioxygen atmosphere, respectively. The presence of 16 electron [(eta5-C5H5)Ru(R,R-dippach)]+ species in fluorobenzene solutions of the corresponding dinitrogen or dioxygen complexes in conjunction with the presence of [BAr'4]- gave in some cases a small fraction of [Ru(eta5-C5H5)(eta6-C6H5F)][BAr'4] (5a), which has been isolated and characterized by X-ray diffraction.     

Activity of an enzyme immobilized on polyelectrolyte multilayers

The immobilization of α-chymotrypsin on the surface of boron silicate glass microspheres is conducted via the technique of multilayer adsorption of polyelectrolytes. It is shown that the enzyme is adsorbed on both positively and negatively charged surfaces and its activity is partially preserved relative to that in solution. The activity of the enzyme depends on the number of polyectrolyte layers preliminarily adsorbed on glass microspheres and on the charge of the surface. The activity of α-chymotrypsin adsorbed on the negatively charged surface is four times higher than the activity of this enzyme adsorbed on a positively charged surface.     

Design of enzyme reactors as chromatographic columns for racemic resolution of amino acid esters

Summary Racemic resolution of aromatic and aliphatic amino acid esters into L-amino acid and D-amino acid ester via LC and HPLC is achieved by using enzyme reactors as chromatographic columns. For this purpose α-chymotrypsin and trypsin are immobilized on Eupergit C, Sepharose 4B and Lichrosorb-Diol.     

The synthesis and thermotropic behaviour of an ethyl cellulose derivative containing azobenzene-based mesogenic moieties

An ethyl cellulose derivative containing azobenzene-based mesogenic moieties was prepared by the reaction of 4-methoxyazobenzene-4′-oxyacetic acid and ethyl cellulose by esterification in the presence of N,N′ -dicylcohexylcarbodiimide and 4-dimethylaminopyridine. Its chemical structure and liquid crystalline properties were characterized by FTIR, ¹H NMR, POM, DSC and WAXD. The degree of substitution of the cellulose backbone by the azobenzene-based mesogenic moieties is 0.9. The polymer is thermotropic and exhibits liquid crystalline behaviour over the temperature range 125–172°C.     

Sequosempervirin A, a novel spirocyclic compound from Sequoia sempervirens

A novel spirocyclic compound (4R)-4-(4-hydroxy-benzyl) spiro [4,5] dec-1-en-8-ol (sequosempervirin A) was isolated from the branches and leaves of Sequoia sempervirens. Its structure and relative stereochemistry were mainly determined by MS, 2D NMR and X-ray means, which is the first naturally occurring norlignan containing one spirocycle with C6 (cyclohexane)-C2-C3-C6 skeleton.     

Does the TauD Enzyme Always Hydroxylate Alkanes, While an Analogous Synthetic Non-Heme Reagent Always Desaturates Them?

This theoretical work addresses the mechanistic switch between hydroxylase (alcohol formation) and desaturase (olefin formation) activities during alkane oxidation by two non-heme high-valent oxoiron reagents, the enzyme taurine:α-ketoglutarase dioxygenase (TauD) and the synthetic shape-selective catalyst (TpOBzFe), toward cyclohexadiene, cyclohexane, cyclopentane, and ethane. As we show, the desaturase/hydroxylase steps obey unique orbital selection rules, and the mechanistic switch is determined by intrinsic reactivity factors that depend on the ligand-sphere flexibility of the oxoiron species, the substrate, and the spin states of the reaction pathways. Testable predictions are outlined.     

Cooperativity and substrate specificity of an alkaline amylase and neopullulanase complex of Micrococcus halobius OR-1

The saccharifying alkaline amylase and neopullulanase complex of Micrococcus halobius OR-1 hydrolyzes both α-(1,4)- and α-(1,6)-glycosidic linkages of different linear and branched polysaccharides. The following observations were made concerning the analysis of the coexpressed amylase and neopullulanase enzymes. Even though the enzymes were subjected to a rigorous purification protocol, the activities could not be separated, because both the enzymes were found to migrate in a single peak. By contrast, two independent bands of amylolytic activity at 70 kDa and pullulanolytic activity at 53 kDa were evident by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), reducing and nonreducing PAGE, and zymographic analysis on different polysaccharides. Preferential chemical modification of the enzyme and concomitant high-performance thin-layer chromatographic analyses of the saccharides liberated showed that amylase is sensitive to 1-(dimethylamino-propyl)-3-ethyl carbodiimide-HCl and cleaved α-(1,4) linkages of starch, amylose, and amylopectin producing predominantly maltotriose. On the other hand, formalin-sensitive neopullulanase acts on both α-(1,4) and α-(1,6) linkages of pullulan and starch with maltotriose and panose as major products. It is understood that neopullulanase exhibits dual activity and acts in synergy with amylase toward the hydrolysis of α-(1,4) linkages, thereby increasing the overall reaction rate; however, such a synergism is not seen in zymograms, in which the enzymes are physically separated during electrophoresis. It is presumed that SDS-protein intercalation dissociated the enzyme complex, without altering the individual active site architecture, with only the synergism lost. The optimum temperature and pH of amylase and neopullulanase were 60°C and 8.0, respectively. The enzymes were found stable in high alkaline pH for 24 h. Therefore, the saccharifying alkaline amylase and neopullulanase of M. halobius OR-1 evolved from tapioca cultivar shows a highly active and unique enzyme complex with several valuable biochemical features.     

Constituents of the pollen of Crocus sativus L. and their tyrosinase inhibitory activity

Five new naturally occurring monoterpenoids, crocusatins-A (1), -B (2a), -C (3), -D (4a) -E (5), a new lactate, sodium (2S)-(O-hydroxyphenyl)lactate (6), and eighteen known compounds were isolated and characterized from the pollen of Crocus sativus L. The tyrosinase inhibitory activities of these compounds were also discussed.     

混合溶剂系统中固定化脂肪酶对酮基布洛芬的催化酯化反应

以硅藻土吸附的脂肪酶为催化剂,对外消旋酮基布洛芬[2-(3-苯甲酰苯基)丙酸]进行对映选择性酯化反应;考察了不同的脂肪酶制剂,固定化时所加缓冲液的体积与pH值,酰基受体(醇)的种类以及混合溶剂系统的组成等因素对酶活性的影响.结果表明,在所考察的7种脂肪酶中,以LipaseOF的酪化活性最高;用硅藻土吸附固定化酶时,缓冲溶液的最适pH为7.0左右,每克酶粉加1.0mL缓冲溶液为最佳;固定化酶催化酯化的活性比游离的脂肪酶高.在酮基布洛芬与不同酰基受体(醇)的酶促酯化反应中,以丙醇的反应速度为最快.在由一种主溶剂与一种助溶剂组成的混合溶剂系统中,酶促酯化的速度要比在单一的主溶剂或助溶剂系统中快.当以1gP值较大的环己烷或异辛烷等为主溶剂,甲苯为助溶剂时,脂肪酶催化酮基布洛芬酯化反应的活性最高.     

Hydrogen transfer to carbonyls and imines from a hydroxycyclopentadienyl ruthenium hydride: evidence for concerted hydride and proton transfer.

Reaction of ([2,5-Ph(2)-3,4-Tol(2)(eta(5)-C(4)CO)](2)H)Ru(2)(CO)(4)(mu-H) (6) with H(2) formed [2,5-Ph(2)-3,4-Tol(2)(eta(5)-C(4)COH)Ru(CO)(2)H] (8), the active species in catalytic carbonyl reductions developed by Shvo. Kinetic studies of the reduction of PhCHO by 8 in THF at -10 degrees C showed second-order kinetics with Delta H(double dagger) = 12.0 kcal mol(-1) and Delta S(double dagger) = -28 eu. The rate of reduction was not accelerated by CF(3)CO(2)H, and was not inhibited by CO. Selective deuteration of the RuH and OH positions in 8 gave individual kinetic isotope effects k(RuH)/k(RuD) = 1.5 +/- 0.2 and k(OH)/k(OD) = 2.2 +/- 0.1 for PhCHO reduction at 0 degrees C. Simultaneous deuteration of both positions in 8 gave a combined kinetic isotope effect of k(OHRuH)/k(ODRuD) = 3.6 +/- 0.3. [2,5-Ph(2)-3,4-Tol(2)(eta(5)-C(4)COSiEt(3))Ru(CO)(2)H] (12) and NEt(4)(+)[2,5-Ph(2)-3,4-Tol(2)(eta(4)-C(4)CO)Ru(CO)(2)H](-) (13) were unreactive toward PhCHO under conditions where facile PhCHO reduction by 8 occurred. PhCOMe was reduced by 8 30 times slower than PhCHO; MeN=CHPh was reduced by 8 26 times faster than PhCHO. Cyclohexene was reduced to cyclohexane by 8 at 80 degrees C only in the presence of H(2.) Concerted transfer of a proton from OH and hydride from Ru of 8 to carbonyls and imines is proposed.     

Dynamic Mechanical Relaxation in the Opaque and Transparent Poly(4-Methyl-1-Pentene) Films The glass transition zone

The thermomechanical properties of opaque and transparent polymer films of a solution of poly(4-methyl-1-pentene) (PMP) in cyclohexane and carbon tetrachloride obtained by casting on teflon and glass plates were investigated. The dynamic mechanical thermal analysis was applied in a frequency range from 0.01 to 100 Hz. The curves of loss tangent vs. temperature varied depending on the sample thermal history. The first part of these curves could relate to the backbone α relaxation into the unperturbed amorphous phase while the next relaxation could result from the backbone α relaxation into amorphous phase perturbed by the presence of the crystal domains. The Arrhenius plots of the first relaxation show a stronger curvature found in each of the transparent samples indicating strong dependency on specific volume. The second one in the case of transparent films and the first one for opaque samples might be approximate to straight lines. This revised version was published online in August 2006 with corrections to the Cover Date.     

The ultra-fast process of picosecond time-resolved energy transfer in liquid cyclohexane by picosecond single-pulse radiolysis

Two formation processes of singlet states of solute molecules in irradiated liquid cyclohexane have been observed by using a single picosecond electron pulse-radiolysis technique. The slower process corresponds to the very fast formation process reported by Beck et al. The faster process is an ultra-fast process which finishes immediately after a picosecond electron pulse, and is hardly quenched by CCl₄ on triethylamine. The ratio of the faster component to the slower component in liquid cyclohexane is very different from that in liquid toluene.     

聚丙烯-环己烷溶液体系的特性粘数-分子量方程

用粘度及凝胶渗透色谱法订定了聚丙烯-环己烷体系60℃的特性粘数方程,[η]=0.123M~0.61。     

Surface functionalization via in situ interaction of plasma-generated free radicals with stable precursor-molecules on cellulose

The surface functionalization process was accomplished in a consecutive 3 step process including: (1) Argon- and oxygen-plasma enhanced generation of free radical sites on cellophane surfaces; (2) “In situ” gas phase derivatization in the absence of plasma using hydrazine, ethylene diamine, or propylene diamine; (3) Second “in situ”, gas phase derivatization in the absence of plasma using oxallyl chloride or “ex situ” derivatization in the presence of glutaraldehyde. The presence of free radical sites on the plasma exposed cellophane surfaces was demonstrated using “in situ” sulfur dioxide and nitric oxide labeling techniques. It was shown that the free radical sites readily react under “in situ” conditions with the stable chain-precursor components and generate the desired spacer-chain molecules. ESCA, ATR-FTIR analysis and dying techniques were used to monitor the cellophane surface changes. A factorial design was used for selecting the optimal plasma parameters. Functionalized cellophane substrates were used for immobilization of α-chymotrypsin in the presence of spacer-chain molecules. The activity of the immobilized α-chymotrypsin was found to be lower in comparison to the activity of the free enzyme and the presence of virgin cellophane in the free enzyme solution also reduced significantly the activity of the enzyme. It is suggested that the swollen state of the cellophane plays a significant role in the decrease of the immobilized enzyme activity.