Enzymatic activity of immobilized enzyme determined by isothermal titration calorimetry

The activity of adsorbed β-glucosidase onto spherical polyelectrolyte brushes (SPBs) is investigated by UV-Vis spectroscopy and isothermal titration calorimetry (ITC). By comparing the results of these two methods, we demonstrate that ITC is a precise method for the study of the activity of immobilized enzymes. The carrier particles used for immobilization here consist of a polystyrene core onto which poly(acrylic acid) chains are grafted. High amounts of enzyme can be immobilized in the brush layer at low ionic strength by the polyelectrolyte-mediated protein adsorption (PMPA). Analysis of the activity of β-glucosidase was done in terms of Michaelis-Menten kinetics. Moreover, the enzymatic activity of immobilized enzyme is studied by ITC using cellobiose as substrate. All data show that ITC is a general method for the study of the activity of immobilized enzymes.     

Characterization of CIM monoliths as enzyme reactors

The immobilization of the enzymes citrate lyase, malate dehydrogenase, isocitrate dehydrogenase and lactate dehydrogenase to CIM monolithic supports was performed. The long-term stability, reproducibility, and linear response range of the immobilized enzyme reactors were investigated along with the determination of the kinetic behavior of the enzymes immobilized on the CIM monoliths. The Michaelis-Menten constant K(m) and the turnover number k(3) of the immobilized enzymes were found to be flow-unaffected. Furthermore, the K(m) values of the soluble and immobilized enzyme were found to be comparable. Both facts indicate the absence of a diffusional limitation in immobilized CIM enzyme reactors.     

Pancreatic enzyme requirements for the dissociation of rat hearts for culture

Summary Nine crude commercially available samples of pancreatic enzyme preparations were examined in an effort to establish enzyme requirements for dissociation of rat heart to single cells for culture. Disc gel electrophoresis, using purified enzymes as references, indicated the presence of at least five enzymes. The levels of these enzymes, trypsin, chymotrypsin, elastase, lipase, and amylase were quantified in the commercial samples by established enzyme assays. The crude enzyme preparations were compared for their abilities to provide good yields of viable cells from the hearts of neonatal white rats. The abilities of the cells to thrive in culture and to beat rhythmically were also used in the comparison. Commercial purified samples of the five enzymes were used singly and in various combinations in preparing cultures to establish minimal pancreatic enzyme requirements for dissociation of rat hearts. It was concluded that at least trypsin, chymotrypsin, and elastase were required to obtain viable rat heart cells in high yield. Amylase and lipase activities were shown not to be necessary for dissociation. Most commercial samples of trypsin, commonly used to dissociate heart tissue, contain trypsin, chymotrypsin, and elastase in concentrations sufficient to release heart cells in good yield with minimal damage. The destruction of cells observed with some of the commercial samples examined was not due to improper levels of trypsin, chymotrypsin, or elastase. Work is in progress to identify the destructive agent(s). This work was supported in part by United States Public Health Service Grant HL10018 and The Pennsylvania State University Agricultural Experiment Station Grant 1858. Authorized for publication on December 28, 1973 as paper 4602 in the journal series of the Pennsylvania Agricultural Experiment Station.     

Clinical uses of enzyme electrode probes

Clinical applications of enzyme electrochemical sensors are reported; they are based on the coupling of enzymes with potentiometric membrane electrodes (pH, iodide) or potentiometric probes (ammonia, carbon dioxide) or amperometric devices (oxygen, hydrogen peroxide). The most popular and successful immobilization procedures for enzymes are reviewed, namely physical entrapments and chemical methods for binding enzymes to solid support like collagen and nylon net; procedures specifically developed for clinical uses of enzyme probes.The simplicity of the apparatus is evidenced, and it is explained how a single instrument can be useful for all kind of measurements. Practical suggestions for constructing a typical probe are given. Single paragraphs are devoted to the determination of urea, cholesterol, creatinine, amino acids, glucose, lactate, protein, choline and acetylcholine to clarify the sequence of enzymatic and electrochemical reactions in order to elucidate the application range the sensitivity and the selectivity as well as the relevant interferences for each metabolite either in the enzymatic or in the electrochemical step. The applications performed in vitro, in vivo and ex vivo and the commercial availability of some instruments are reported.     

Evaluation of methods for the determination of mitochondrial respiratory chain enzyme activities in human skeletal muscle samples

The quantification of mitochondrial enzyme activities in skeletal muscle samples of patients suspected of having mitochondrial myopathies is problematic. Therefore, we have evaluated different methods for the determination of activities cytochrome c oxidase and NADH:CoQ oxidoreductase in human skeletal muscle samples. The measurement of cytochrome c oxidase activity in the presence of 200 microM ferrocytochrome c and the detection of NADH:CoQ oxidoreductase as rotenone-sensitive NADH:CoQ(1) reductase resulted in comparable citrate synthase-normalized respiratory chain enzyme activities of both isolated mitochondria and homogenates from control human skeletal muscle samples. These methods allowed the precise detection of deficiencies of respiratory chain enzymes in skeletal muscle of two patients harboring only 20 and 27% of deleted mitochondrial DNA, respectively. Therefore, citrate synthase-normalized respiratory chain activities can serve as stable reference values for the determination of a putative mitochondrial defect in human skeletal muscle.     

Quantification of enzyme activities in brain sections by microphotometry

1. Catalytic enzyme histochemistry offers the possibility to demonstrate enzyme activities quantitatively (microphotometry) in brain sections of those sites where they are localized. 2. A prerequisite for quantification are appropriate histochemical procedures for the demonstration of enzymes, which are shortly discussed. 3. For the microphotometric determination of enzymes in brain sections the scanning microphotometry is at present the technique of choice. 4. This is described in the example of an image plane scanning system. 5. Using this technique two measuring procedures can be applied for the quantification of enzyme activities, i.e. kinetic and end-point measurements. 6. Methods for the microphotometric determination of certain important oxido-reductases and further enzymes are presented. 7. It is concluded that quantitative catalytic enzyme histochemistry could be a source of results complementary to those provided by conventional biochemistry.     

Effect of proteolytic enzyme on dyeing of wool with madder

In recent years, the use of low-environmental impact biotechnology giving rises to new types of treatment in the textile industry. The use of protease enzymes to improve some physical and mechanical properties such as smoothness, drapeability, dyeing affinity and water absorbency is particularly interesting. In this research, wool yarns were first treated with different concentrations of protease enzymes in water solution including 1, 2, 4 and 6% o.w.f. for 60 min. The dyeing process was then carried out on the treated yarns with madder (50% o.w.f.). Tensile strength of treated yarns was decreased due to enzyme treatment and it continued to decrease with an increase in enzyme concentration in solution. The L^* values decreased for the samples treated with enzyme. The wash and light fastness properties of samples were measured according to ISO 105-CO5 and Daylight ISO 105-BO1. The washing fastness properties of treated samples were not changed. In the case of light fastness properties, it was increased a little for 4% and 6% enzyme treated samples.     

Determination of phosphate Ions with an enzyme sensor system

An enzyme sensor system for the determination of phosphate ions was constructed using immobilized enzymes and an oxygen electrode. The principle of this method is based upon the nucleoside phosphorylase catalyzed reaction for which the presence of inorganic phosphorus is indispensable. One assay could be completed within 3 min. This enzyme sensor was able to withstand at least 70 assays. This system was applicable to simple, rapid and continuous determination of phosphate ions in food.     

Screen-printed enzyme sensors for l-lysine determination

Sensors for the determination of L-lysine in samples of fermentation broth have been developed. Low-cost screen-printed sensors comprising a platinum working electrode, an Ag/AgCl pseudo reference and a carbon counter electrode were used as transducers for the enzyme sensors. L-lysine-(alpha)-oxidase from Trichoderma viride has been immobilized by entrapment into a polyurethane hydrogel. Sensors were characterized for L-lysine with respect to pH value, linear range, reproducibility, repeatability, storage and working stability. The sensitivities to other amino acids were also determined. A batch system with two working electrodes, one with immobilized enzyme and one without was adapted for the determination of L-lysine by differential measurements. Good agreement was found between L-lysine concentrations measured by the enzyme sensors and by a conventional amino acid analyzer.     

Evaluation of the enzyme thermistor as a specific detector for chromatographic procedures

The successful application of a simple flow calorimeter, called the enzyme thermistor, to the specific monitoring of different enzymes in the eluants from some common chromatographic procedures is described. The enzyme thermistor enables a specific enzyme to be continuously determined even in optically dense solutions where spectrophotometric procedures cannot be used. The instrument can be operated either on-line or it can be used for discrete samples. The sensitivity is in the order of 0.1 IU/ml for most enzymes.     

Immobilization of cellulase on magnetoresponsive graphene nano-supports

In this study, we report the preparation of pH tunable, temperature sensitive magnetoresponsive graphene-based nano-bio carriers for cellulase immobilization. We discuss a simple route to overcome the geometric disadvantage imposed by most 2D immobilization supports and make them capable of closely mimicking free enzymes (FE) operating under similar reaction conditions. The supramolecular assembly of oppositely charged quenched polyelectrolytes and maghemite–magnetite nanoparticles on 2D graphene supports followed by covalent immobilization of cellulase shows a marked improvement in the bio-receptivity of graphene supports. The incorporation of magnetic nanoparticles opens up the possibility of recovery and reuse of the enzyme over multiple cycles. The immobilized enzymes retained about 55% of the original specific activity even after four cycles of reuse. Cellulase immobilization is achieved by a combination of annealed polyelectrolyte brushes and zero-length spacer molecules. The swelling behavior of annealed polyelectrolyte brushes is a strong function of the environmental conditions. The degree of polyelectrolyte swelling can be easily tweaked by manipulating the pH and temperature, providing us an effective tool to control the activity of immobilized enzymes. At a pH of 5.1 and a temperature of 50 °C, the immobilized enzymes with the annealed polyelectrolyte brushes displayed close to 1.5-fold improvement in the activity as compared to immobilized enzymes without the brushes. Activity of immobilized cellulase is evaluated using both soluble as well as insoluble substrates like 2% (w/v) CMC and avicel respectively.     

Enzyme family-specific and activity-based screening of chemical libraries using enzyme microarrays

The potential of protein microarrays in high-throughput screening (HTS) still remains largely unfulfilled, essentially because of the difficulty of extracting meaningful, quantitative data from such experiments. In the particular case of enzyme microarrays, low-molecular-weight fluorescent affinity labels (FALs) can function as ideally suited activity probes of the microarrayed enzymes. FALs form covalent bonds with enzymes in an activity-dependent manner and therefore can be used to characterize enzyme activity at each enzyme's address, as predetermined by the microarraying process. Relying on this principle, we introduce herein thematic enzyme microarrays (TEMA). In a kinetic setup we used TEMAs to determine the full set of kinetic constants and the reaction mechanism between the microarrayed enzymes (the theme of the microarray) and a family-wide FAL. Based on this kinetic understanding, in an HTS setup we established the practical and theoretical methodology for quantitative, multiplexed determination of the inhibition profile of compounds from a chemical library against each microarrayed enzyme. Finally, in a validation setup, K(i)(app) values and inhibitor profiles were confirmed and refined.     

Characterization of soil samples by enzyme activity

The presence of extracellular enzymes in soils offers a very sensitive method for matching, or distinguishing between, soil samples. Simple colorimetric methods were used to compare enzyme levels in soils from different sites. It was found that each soil tested had its own spectrum of enzyme activity, by which it could be characterized and thereby distinguished from other soils that were essentially similar in physical composition.

This article describes nine simple enzyme assays used by sixth-form students for ‘soilprinting’, and draws attention to some possible applications of the technique in forensic science and in studies of soil fertility.     

The presence of desulfo xanthine dehydrogenase in purified and crude enzyme preparations from rat liver

Crude and purified xanthine dehydrogenase preparations from rat liver were examined for the existence of a naturally occurring inactive form. Reduction of the purified enzyme by xanthine under anaerobic conditions proceeded in two phases. The enzyme was inactivated by cyanide, which caused the release of a sulfur atom from the molybdenum center as thiocyanate. The amount of thiocyanate released was almost in parallel with the initial specific activity. The active and inactive enzymes could be resolved by affinity chromatography on Sepharose 4B/folate gel. These results provided evidence that the purified enzyme preparation from rat liver contained an inactive form. A method for the determination of the active and inactive enzymes in crude enzyme preparations from rat liver was devised based on the fact that only active enzyme could react with [14C]allopurinol and both active and inactive enzymes could be immunoprecipitated quantitatively by excess specific antibody to xanthine dehydrogenase. The amount of [14C]alloxanthine (derived from [14C]allopurinol) bound to the active sulfo enzyme in crude rat liver extracts was about 0.5 mol/mol of FAD. As this content is closely similar to that in the purified enzyme, these results suggest the existence of an inactive desulfo form in vivo.     

A rapid method for the determination of proteolytic activities of enzyme preparations

A method has been described for the determination of proteolytic activities of enzyme preparations using casein as substrate. The rate of digestion is proportional to the enzyme concentration used. This relationship is utilized as a measure of the enzyme activity. One unit of activity is defined as the amount which is required to digest casein in 15 minutes at 37.5 degrees C. so that 50 per cent of the protein in 1 ml. of 0.25 per cent solution is not precipitable by trichloroacetic acid. This method has been used to determine the activity of enzymes from different sources and also used to follow the rate of activation of enzymes.     

Plasma volume determination by use of enzyme dilution in the dog

The use of enzymes for plasma volume determination in the dog was investigated. Plasma volume was determined from dilution of intravenously injected enzymes and compared with results obtained by high molecular weight fluorescein-labelled dextran. Aspartate aminotransferase and alanine aminotransferase from liver tissue gave good results, but the activities of creatine kinase and lactate dehydrogenase from heart tissue were inhibited in plasma, resulting in an overestimation of plasma volume. Enzymes offer a useful alternative to methods presently used since no radioactive isotopes are needed. In laboratory animals the enzyme preparations are readily obtainable and there is minimal risk of immunological complications arising after repeated determinations.     

Crystallization of an NADP+-dependent malic enzyme from rat liver

Crystals of a tetrameric NADP+-dependent malic enzyme from rat liver have been grown in the presence of NADP+ using the hanging-drop method of vapour diffusion with ammonium sulphate as the precipitant. Measurement of the crystal density and calculation of the values of Vm for different numbers of polypeptide chains in the unit cell indicate that the asymmetric unit of the crystal contains a complete tetramer, allowing the application of non-crystallographic symmetry to the determination of the molecular structure of this enzyme. This structure would provide only the second example for an enzyme involved in oxidative decarboxylation, the other being 6-phosphogluconate dehydrogenase. In addition, then, to providing an insight into the structure-function relationship in malic enzyme, the successful structure determination would permit valuable comparisons to be made between these two and other enzymes with this catalytic activity.     

High-performance liquid chromatography determination of enzyme activities in the presence of small amounts of product

Enzymes can be assayed by HPLC by calculating the amount of substrate(s) left over, or product formed, through the peak area ratios with a suitable internal standard. However, sometimes the substrates used are contaminated with small amounts of products and this can lead to errors in the determination of the enzyme activity. A method for a HPLC test of such enzymes, which prevents eventual errors, uses the ratio substrate/product at time zero as internal standard and the kinetics can be followed with the aid of a simple mathematical equation. This approach was applied to the determination of the activities of papain, urokinase, NAD glycohydrolase, and pyruvate kinase samples and it was compared with the data obtained by the internal standard method, giving reproducible results in all cases.