The effect of sorbitol on acid phosphatase deactivation

Acid phosphatase thermal deactivation follows a complex path: an initial decay toward an equilibrium distribution of at least two intermediate structures, mutually at the equilibrium, followed by a final breakdown toward a completely inactive enzyme configuration. The results obtained in the presence of sorbitol have been compared to those produced in the course of purely thermal deactivation of the native enzyme. For any sobitol concentration, an equivalent temperature is calculated that results in exactly the same activity-versus-time profile. This suggests enzyme deactivation to be controlled by a single, unchanging step. Immobilized enzyme runs have been performed, as well, by entrapping acid phosphates within a polymeric network formed onto the upstream surface of an ultrafiltration membrane. The stabilizing effect of entrapment cumulates with that produced by sorbitol. In this case, however, an equivalent temperature cannot be determined, thus indicating that a different deactivation mechanism is followed.     

Solid-state enzyme deactivation in air and in organic solvents

Thermal deactivation of solid-state acid phosphates (E.C. 3.1.3.2, from potato) is analyzed, both in the presence and in the absence of organic solvents. The thermal deactivation profile departs from first order kinetics and shows an unusual activity. The process is described by a phenomenological equation, whose theoretical implications are also discussed. The total amount of buffer salts in the enzyme powder dramatically affects enzyme stability in the range 70xC to 105xC. The higher salt/protein ratio increases the rate of thermal deactivation. The deactivation rate is virtually unaffected by the presence of organic solvents, independent of their hydrophilicity. (c) 1994 John Wiley & Sons, Inc.     

A note on the use of urea in studying the mechanism of thermal inactivation of extracellular proteinase from pseudomonas fluorescens 22F

Strong denaturants can be used to distinguish between heat-induced changes in the primary structure of the enzyme molecule and heat-induced changes in higher orders of structure. In this paper, we report on an attempt to use urea in studying the mechanism of thermal inactivation of the extracellular proteinase from Pseudomonas fluorescens 22F. Addition of urea at> 2 (without heating) resulted in inactivation which was, however, reversible. Diluting to concentrations < 2 urea completely restored proteolytic activity. The rate of inactivation at 100°C of the proteinase was increased when 6 urea was present during heat treatment. Also at lower urea concentrations, the inactivation rate at 100°C was increased. Addition of 6 urea to the enzyme solution after heat treatment also increased the extent of inactivation while low urea concentrations (< 1 ) did not. It was concluded that cyanate formed from urea at high temperature was the cause of increased inactivation since addition of cyanate could increase the inactivation rate while a treatment to remove cyanate from a heated urea solution could prevent increase tnactivation. The use of urea does not appear to be suitable for the elucidation of the mechanism of thermal inactivation of the extracellular proteinase from P. fluorescens 22F, but might be applicable to other enzymes when treated (cyanate free) urea is used after heat treatment; however, use of urea (even if cyanate free) during heat treatment is not possible because cyanate is induced by the very heat treatment.     

Strategies in the Preparation of Homochiral Compounds Using Combined Enantioselective Enzymes

In order to obtain a homochiral product from a racemic substrate, different strategies can be followed using a moderately enantioselective enzymatic catalyst. Two new strategies are presented, involving the simultaneous use of two enzymes, parallel or consecutive. In the parallel system, the substrate enantiomer yielding the unwanted product enantiomer is enantioselectively converted by the second enzyme. In the consecutive system, the substrate enantiomer yielding the desired product enantiomer is itself the preferred product of another enantioselective enzymatic reaction.

For irreversible pseudo-first order enzyme kinetics, a relationship was found which describes the dependency of the yield and enantiomeric excess for these systems on the E-values of the separate enzymes and on the ratio of their concentrations. For Michaelis-Menten kinetics, these relationships usually give good approximations.

According to these calculations, the yield and enantiomeric excess obtainable with the concepts of combined enzymes exceed significantly those obtainable with the separate enzymes, and also those obtainable with the strategy of product recirculation.     

The Influence of Fatty Acid and Glycerol on the Kinetics of Fat Hydrolysis by Candida Rugosa Lipase in a Membrane Reactor

The kinetics of lipid-hydrolysis by Candida rugosa lipase was investigated in a membrane reactor and in an emulsion system. Two models were chosen to describe the kinetics of the enzyme:

(1) The hydrolysis of triglycerides to fatty acids was considered to be a chain reaction with the intermediary products di- and mono-glyceride; each step was assumed to be a reversible second-order reaction. The reaction rate constants were determined from batch experiments. The experimental results could be described with this model.

(2) For process optimization and control, a model based on the power law was developed. For this model, the rate of hydrolysis was measured as a function of fatty acid and glycerol concentrations. Relations for the initial rate and equilibrium ester fraction as a function of the glycerol concentration were determined. Further, the reaction rate could be described with the power-law model with a power of 1.75 in the hydrolyzable ester fraction for a wide range of glycerol concentrations. The model with power 1.75 gave much better results when compared to a similar first order model. Although simpler, the first order model can not be used. The power law model was applied in the simulation of a reactor composed of three modules. The fatty acid production rate was calculated for this reactor system as a function of the outgoing glycerol concentration at different conditions.     

Series mechanism of enzyme deactivation. Characterization of intermediate forms

Acid phosphatase (E.C. 3.1.3.2) undergoes complex thermal deactivation phenomena, as revealed by the two-slope pattern of the enzyme logarithmic-specific-activity versus time curves. The native enzyme first decays toward an equilibrium distribution of less, but still active, intermediate structures and these, in turn, undergo a final degradation to a completely inactive form. The effect of the experimental conditions at which the enzyme is kept during the deactivation process on the characteristics of these intermediate enzymatic structures has been investigated. The kinetic parameters of p-nitro-phenyl phosphate hydrolysis, as catalyzed by some of these intermediate forms, have been determined and the results compared to those obtained with the native enzyme.     

Carboxypeptidase Y-Catalyzed Reaction in Systems Containing Organic Solvent

The effect of organic solvents on carboxypeptidase Y (a serine carboxypeptidase from yeast)-catalyzed hydrolysis of amino acid ester and peptide synthesis from N-acyl amino acid ester and amino acid amide was investigated.

The K_m value of ester hydrolysis increased with an increase in the solvent content. Dioxane was the most effective and dimethyl sulfoxide (DMSO) the least, whilst K_cat showed a tendency to increase slightly in N, N-dimethylformamide (DMF) and DMSO. For dioxane and acetonitrile (MeCN) a maximum was observed.

In peptide formation from Fua-Phe-OEt and Gly-NH₂, dioxane and MeCN supported high product yield at molar fractions smaller than ca. 0.05 but the yield decreased significantly at higher fractions, although a relatively constant selectivity (ratio of the peptide bond formed to the ester consumed) was maintained. DMSO gave rather low peptide yields and selectivity even at lower molar fractions. DMF showed an intermediate tendency.

An apparent saturation parameter of the amine component was evaluated and the dissociation constant of a complex between acyl-enzyme and amino acid amide (K_n), as well as the rate constant of aminolysis exerted by the amino acid amide bound correctly on the enzyme (K_n), was calculated by initial rate analysis of peptide formation. In contrast to K_m values, K_n decreased with increasing concentrations of organic cosolvent. while a suppressive effect was observed (except for DMSO) on the K_n parameter.

Effects of the solvent practically immiscible in water was also studied by use of the enzyme physically “immobilized” on glass beads.     

Enzyme stability in the presence of organic solvents

Summary The thermal stability of vacuum-dried acid-phosphatase has been investigated, both in the absence and in the presence of pure hexadecane. Preliminary experimental results indicate that: i) in both solid-phase runs, acid-phosphatase is much more stable than the free enzyme in aqueous solution, ii) the presence of the organic solvent slightly reduces thermal stability of the solid-phase enzyme. As regards the deactivation mechanism, when acid-phosphatase operates in free aqueous solution it follows a two-step in series deactivation. Initially the native configuration decays towards an intermediate, still active form. This, in turn, irreversibily yields a totally inactive structure. In the thermal deactivation of solid-phase enzyme it has been observed that: i) the first step is substantially retarded, ii) the final transition is completely hindered, iii) the intermediate configuration is more active than that produced in aqueous solution, by more than one order of magnitude.     

Kinetic inactivation study of mushroom tyrosinase: intermediate detection by denaturants

The unfolding and inhibition study of mushroom tyrosinase have been studied in the presence of different denaturants such as sodium dodecyl sulfate (SDS), guanidine hydrochloride (GdnHCl), and urea. The kinetic two-phase rate constants were commonly measured from semilogarithmic plots of the activity versus time, which resolved into two straight lines, indicating that the inactivation process consisted of fast and slow phases as a first-order reaction. This result also implied that transient partially folded intermediate existed during tyrosinase unfolding pathway. Mushroom tyrosinase had different behaviors to denaturants in regard with: noncooperative binding manner by SDS while cooperative interactions by GdnHCl and urea; in equilibrium state, SDS-micelle never completely inactivated enzyme activity while GdnHCl has single step denaturation and urea induced a typical transition-like process. Various kinetic parameters for each denaturant were calculated and the possible unfolding pathway scheme was discussed.     

Effect of Additives on the Inactivation of Lysozyme Mediated by Free Radicals Produced in the Thermolysis of 2, 2-Azo-Bis-(2-Amidinopropane)

The inactivation of lysozyme caused by the radicals produced by thermolysis of 2, 2-azo-bis-2-amidino-propane can be prevented by the addition of different compounds that can react with the damaging free radicals. Compounds of high reactivity (propyl gallate, Trolox, cysteine, albumin, ascorbate, and NADH) afford almost total protection until their consumption, resulting in well-defined induction times. The number of radicals trapped by each additive molecule consumed ranges from 3 (propyl gallate) to 0.12 (cysteine). This last value is indicative of chain oxidation of the inhibitor. Uric acid is able to trap nearly 2.2 radicals per added molecule, but even at large (200 μM) concentrations, a residual inactivation of the enzyme is observed, which may be caused by urate-derived radicals.

Compounds of lower reactivity (tryptophan, Tempol, hydroquinone, desferrioxamine, diethylhydroxylamine, methionine, histidine, NAD⁺ and tyrosine) only partially decrease the lysozyme inactivation rates. For these compounds, we calculated the concentration necessary to reduce the enzyme inactivation rate to one half of that observed in the absence of additives. These concentrations range from 9 μM (tryptophan and Tempol) to 5 mM (NAD⁺).     

Acid Versus Neutral Formalin Solution as a Neurological Fixative

The fixing action of 10% formalin solution alone and with formic, acetic, propionic, butyric, lactic, monochloracetic, dichloracetic, or trichloracetic acid was studied by means of stains with silver, osmic acid and cresyl violet. The following conclusions were reached:

1. In general, better fixation and staining was obtained with acid than without.

2. Less difference was seen in comparing one acid with another than was expected before the experiments were made.

3. Propionic, butyric, and dichloracetic showed no promise of having practical value.

4. Formic and monochloracetic acids as modifiers gave superior stains with osmic acid, while silver and cresyl violet stains of the same material were about equal to those made from formalin-acetic fixed material.

5. Lactic acid caused somewhat more distortion of tissue elements than the others but was compatible with good staining.

6. Acetic acid was most effective in concentrations of 3 to 5% while the stronger acids such as formic, monochloracetic, lactic and trichloracetic were effective in concentrations of 0.5 to 1%.     

Effect of Water Diffusion Limitations on the Thermostability of Enzymes in Non-Aqueous Environments

The thermostability of anhydrous α-chymotrypsin has been analysed both in air and in organic solvents, with regard to the effect of the protein water-content on the course of deactivation. A higher initial water content increases the rate of inactivation.

Deactivation tests carried out under a constant thermodynamic activity of water indicate that reductions in dehydration rate lead to lower stability.

The effect of water diffusion phenomena has also been studied. Protein aggregates of larger size are less thermostable, thus indicating that diffusional limitations to water transfer can play a significant role in thermoinactivation.

The effect of water content on enzyme thermostability was also measured in the presence of two organic solvents of different hydrophobicity. In both cases, the resulting increased thermolability can be explained in terms of a limitation in water transfer towards the non-aqueous environment.     

Use of isoelectric focusing and a chromophoric organomercurial to monitor urea-induced conformational changes of yeast phosphoglycerate kinase.

The effects of urea in concentrations from 0 to 6M on the following properties of yeast phosphoglycerate kinase were studied: the kinetics of inactivation of the enzyme, the spectrum of 2-chloromercuri-4-nitrophenol bound to the single thiol group of the enzyme, the rate of reaction between the mercurial and enzyme, and the isoelectric point. The enzyme was inactivated by as much as 30% in 1M-urea, and the other data were interpreted as a possible 'tightening' of enzyme structure. The catalytic behaviour of the enzyme in 2M-urea was time-dependent, the initial effects being similar to those in 1M-urea. Polyacrylamide-gel isoelectric focusing of the enzyme in the presence of 2M-urea showed a single species of enzyme with an isoelectric point intermediate between those in 1M- and 3M-urea; a species with an identical isoelectric point was obtained after an 11-day exposure at 4 degrees C to the denaturant at 2M. The enzyme was rapidly inactivated in 3M-urea, with the thiol group fully exposed and the isoelectric point 0.9pH unit higher than in the absence of urea. No further conformational changes could be demonstrated with urea concentrations of 4M or greater. It is suggested that the equilibrium species that exists in 2M-urea has one of two buried lysine residues exposed. The second lysine residue is exposed in 3M or greater concentrations of the denaturant.     

Quantifying the inactivation rate constants for the molecular species comprising the catalytic cycle of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase

When an unstable enzyme is incubated with its substrate(s), catalysis may cease before chemical equilibrium is attained. The residual substrate concentrations depend on their initial concentrations, the initial enzymic activity, and the inactivation rate constants for each molecular species that comprise the catalytic cycle. The underlying theory has been elaborated previously for single-substrate reactions and here it is extended to bi-substrate reactions. The theory is illustrated by application to glucose 6-phosphate dehydrogenase, which is unstable when exposed to a low concentration of sodium dodecyl sulphate. It is shown that the ternary complex containing both substrates is resistant to inactivation while each of the remaining complexes undergoes first-order decay. Rate constants for the inactivation of each complex are calculated.     

A Bioreactor-Crystallizer for L-Malic Acid Production

A bioreactor with associated crystallizer for the accumulation of a highly concentrated slurry product has been developed and investigated. The transformation of Ca-fumarate to Ca-L-malate by the action of the fumarase of immobilized Brevibacterium flavum cells focussed on the performance of this newly-devised bioreactor-crystallizer system.

The following results were obtained

(1) The fumarase reaction in the bioreactor proceeded at a rate that was first-order in apparent substrate concentration.

(2) The reaction rate increased with the addition of Na₂-fumarate to the substrate solution.

(3) The reaction rate was independent of the substrate circulation rate and the initial substrate concentration in the crystallizer.

(4) Fumarase activity of immobilized B. flavum cells was stable after 10 repeated uses over a period of 10 days.

(5) Maximum concentration of the product, final conversion ratio of the substrate and the productivity of the bioreactor-crystallizer system were much higher than those for a conventional bioreactor using solubilized Ca-fumarate as a substrate.     

Role of mitochondrial oxidative stress to explain the different longevity between genders: protective effect of estrogens

Females live longer than males. Work from our laboratory has shown that this may be due to the up-regulation of longevity-associated genes by estrogens. Estrogens bind to the estrogen receptors and subsequently activate the mitogen activated protein kinase and nuclear factor kappa B signalling pathways, resulting in an up-regulation of antioxidant enzymes.

Estrogen administration, however, has serious undesirable effects and of course, cannot be administered to males because of its powerful feminizing effects. Thus, we tested the effect of genistein, a phytoestrogen of high nutritional importance whose structure is similar to estradiol, on the regulation of the expression of antioxidant, longevity-related genes and consequently on oxidant levels in mammary gland tumour cells in culture. Phytoestrogens mimic the protective effect of oestradiol using the same signalling pathway.

The critical importance of up-regulating antioxidant genes, by hormonal and dietary manipulations, to increase longevity is discussed.     

Effect of Daunorubicin on Subcellular Pools of Glutathione in Cultured Heart Cells from Neonatal Rats

Alterations in cellular GSH and its compartmentation were investigated as a possible mechanism of toxicity of the anthracycline derivative daunorubicin in neonatal heart cells. Cultured beating heart cells from neonatal rats were exposed to daunorubicin at therapeutically relevant concentrations and the resulting changes in cellular GSH as well as cytosolic and mitochondrial pools of GSH were determined. Toxicity was estimated as an increased permeability of the plasma membrane to cytosolic enzymes, e.g., lactate dehydrogenase.

Control heart cells were found to contain 12.2 ± 1.8 nmolesGSH/IO⁶ cells. Daunorubicin causedarapid initial decrease followed by a transient increase in cellular GSH. The extent of the latter increase was dependent on the concentration of daunorubicin. High concentrations of daunorubicin gave only a slight increase followed by a pronounced decrease in cellular GSH.

By applying a digitonin-based method the effect of daunorubicin on the cytosolic and mitochondrial pools of GSH were separated. The concentration of cytosolic and mitochondrial reduced GSH was estimated to be 89 ± 1.5nmoles, 10 cells and 3.3 ± 0.6 nmoles/10⁶ cells. respectively. The results indicate that daunorubicin caused a decrease of cytosolic GSH and. after a short lag period. a release of lactate dehydrogenase. No decrease of mitochondrial GSH occurred under these conditions indicating that daunorubicin influences selectively cytosolic GSH.

No lipid peroxidation products were detected in DRB-treated cells under conditions when lactate dehydrogenase was released. Likewise, addition of the iron-chelator desferrioxamin did not influence the release of lactate dehydrogenase. whereas dithiothreitol offered partial protection.

The results provide support for an oxidative mechanism in which the decrease in the cytosolic pool of GSH may be the causative factor of daunorubicin-induced toxicity. This decrease in GSH may affect the cytosolic NADPH and various redox groups on proteins, thereby altering the permeability of the plasma membrane and finally causing cell damage.     

Immobilization of lipase from Candida rugosa on Eupergit C supports by covalent attachment

The present study compares the results of three different covalent immobilization methods employed for immobilization of lipase from Candida rugosa on Eupergit^® C supports with respect to enzyme loadings, activities and coupling yields. It seems that method yielding the highest activity retention of 43.3% is based on coupling lipase via its carbohydrate moiety previously modified by periodate oxidation. Study of thermal deactivation kinetics at three temperatures (37, 50 and 75 °C) revealed that the immobilization method also produces an appreciable stabilization of the biocatalyst, changing its thermal deactivation profile. By comparison of the t_1/2 values obtained at 75 °C, it can be concluded that the lipase immobilized via carbohydrate moiety was almost 2-fold more stable than conventionally immobilized one and 18-fold than free lipase. The immobilization procedure developed is quite simple, and easily reproduced, and provides a promising solution for application of lipase in aqueous and microaqueous reaction system.     

Phase II Trial of Copper Zinc Superoxide Dismutase (CuZn SOD) in the Treatment of Crohn'S Disease

Bovine Cu Zn SOD was used during an 8 year period as an antiinflammatory drug in 26 patients with severe Crohn's disease (CDAI 300) usually after failure of corticotherapy or when this drug was discontinued because of side eKects or infection.

This was a phase II trial during which doses routes of administration and concomitant therapies were progressively modified.

We obtained 73% good short term responses (judged upon CDAI and anatomic healing) and 82% positive results on long term evolution (the criteria were : i CDAI lower than 100 in between relapses, ii complete healing or notable improvement of lesions, iii no surgery needed, iv return to work. The acceptability was excellent with the free enzyme. Since the above described experience, published in Free Radical Biology and Medicine (1989, 7 : 145-151). we used always the same treatment schedule (SOD 8 mg/day associated with Desferroxamine -500 mg subcutaneous every 2 days). The follow-up during the 87-89 period showed that 12 are in good health without any relapse, 9 experienced one or more relapses, and showed good responses upon resumption of treatment, 5 failed to respond to treatment. all part of the initial group on which SOD treatment had already failed, and among whom 3 were lost for follow-up before 1987, and two others took up another SOD treatment which also failed. 3 new patients (2 females, 1 male) were treated since then, and all 3 had positive results (one with disappearance of ileococcal mass).

The efficacy of SOD as an antiinflammatory drug in Crohn's disease needs to be confirmed by controlled trials.     

Non-Enzymic Lipid Peroxidation in Microsomes and Microsomal Phospholipids Induced by Anthracyclines

The stimulation of non-enzymic lipid peroxidation by doxorubicin, daunorubicin and 7 derivatives was investigated in extracted microsomal phospholipids and in intact microsomes.

Evidence was obtained for the necessity of a free amino-sugar moiety for a stimulative effect on lipid peroxidation. Binding of anthracyclines to RNA (which is present in microsomes) was inhibitory towards stimulation.

Drugs that stimulated lipid peroxidation in a non-enzymic system with extracted phospholipids also were stimulative in an enzymic, NADPH-dependent, microsomal system. They were not always effective in intact microsomes without the enzymic system.

The role of the enzymic system in the stimulation of anthracycline induced lipid peroxidation is thought to be the reduction of iron ions rather than the stimulation of oxygen radical production via the anthracyclines.