A semiautomated system for measurement of 96 simultaneous spectrophotometric enzyme assays

A semiautomated system for spectrophotometric measurement of enzyme activity is described. In comparison to a 1-ml reaction volume monitored continuously by a conventional spectrophotometer, this system requires 1/10 to 1/100 the volume of sample, and 1/8 to 1/4 the time for measurement and computation of 96 enzyme assays. The system hardware consists of a 96-well platereader interfaced to a personal computer. Absorbances of 96 reactions are measured at timed intervals. These data are transmitted electronically from the platereader to the computer through the modem port using a modem program. The reaction rates are computed from the timed absorbance readings using a spreadsheet program. Three enzyme assays are presented, but the method has been used for several other assays and is applicable to many spectrophotometric rate assays. Many laboratories currently possess one or both of the two major components of the relatively inexpensive system described.     

Factor-VIII-related antigen: measurement by enzyme immunoassay.

Factor-VIII-related antigen was measured, both by an enzyme immunoassay using a microplate method and by the Laurell technique, in normal people, patients with von Willebrand''s disease, haemophiliacs, and obligatory haemophilia carriers. The enzyme immunoassay was simpler to perform and gave equally reliable and reproducible results. Many more assays could be carried out at any one time.     

Validation of an enzyme immunoassay for measurement of excreted estrogen and testosterone metabolites in the white-crowned sparrow (Zonotrichia leucophrys oriantha)

Over the past few years there has been a growing interest in hormonal analyses, despite the cost and difficulty involved in measuring hormones using radioimmunoassays. Therefore, there is a need for alternate procedures, such as enzyme immunoassays, capable of providing similar data to that provided by radioactive methods. Enzyme immunoassays are a less expensive method of measuring estrone conjugates and testosterone. With further work, the enzyme immunoassay also provides the potential to adapt the enzyme assay for use in the field. The data in this study demonstrate that an enzyme assay can accurately detect both endogenous and exogenous hormonal changes in the droppings of white-crowned sparrows. © 1995 Wiley-Liss, Inc.     

Improvement of the lectin-antibody enzyme immunoassay of the alphafetoprotein carbohydrate chain for automation with the enzyme immunoassay robot

The lectin-antibody enzyme immunoassay of the alphafetoprotein-L3 carbohydrate chain, a tumor marker of liver cancer, has not been automated. We improved the technique of the assay for automation. Consequently, alphafetoprotein-L3 and total alphafetoprotein were detected with two lectins using an automatic paramagnetic bead handling robot. This indicates that the improved method is potentially applicable to the automated enzyme immunoassay robot.     

An enzyme electrode for measurement of penicillin in fermentation broth: A step toward the application of enzyme electrodes in fermentation control

A penicillin sensitive enzyme electrode has been used to analyze the concentration of benzylpenicillin in fermentation broth. The electrode response time was in the region of 2 min and the response to penicillin concentration was linear within the range of 1 to 10mM. The buffering capacity of the medium influenced the sensitivity of the electrode. At low buffer capacity the sensitivity of the enzyme electrode to penicillin was very high, but then the sensitivity to small changes in buffer capacity was relatively large. At high buffer capacity the sensitivity to penicillin was reduced and the electrode became less dependent on changes to buffer capacity. Constant calibration curves were repeatedly obtained with the electrode when used for 2 hr daily in a fermentation medium over a six day period. Three methods devised to calibrate the electrode for use in fermentation media were investigated. Methods one and two, based on the relationship between electrode sensitivity and buffer capacity in phosphate buffer and in sterile media, gave rather high penicillin concentration values. The third method based on an internal standard was the most satisfactory.     

An enzyme electrode for amperometric measurement of D-amino acid

A carbon paste enzyme electrode has been developed for measurement of D-amino acids that employs a fatty acid modified FAD to prevent leaching of this essential cofactor to the surrounding aqueous environment and which serves as an enzyme stabilizing agent. The lower limit of detection is at least 10(-4) M and the electrode has a linear range of 10(-4) to 3 x 10(-3) M and a response time of 180 s. Twenty L-amino acids were tested and none of them elicited responses when electrodes were exposed to 0.5 mM concentration increases over a baseline level. On the other hand, some response was observed when exposed to 18 of 20 D-amino acids varying from 2 to 200% of the corresponding D-alanine response. Electrodes were shown to have longevities of over 30 days while maintaining 85% of their original sensitivity. Electrodes showed activity over a pH of 6.2-11.7 with a maximum at 9.2 and over temperatures of 10-47 degrees C with a maximum at 37 degrees C.     

A modified ferrozine method for the measurement of enzyme-bound iron

A general procedure for the determination of the iron content of enzymes by digestion with methanesulfonic acid to release protein-bound iron has been developed. This procedure replaces the tedious and potentially hazardous method of wet ashing with concentrated nitric-sulfuric-perchloric acids. The method has been used to determine the stoichiometry of iron for nanomole quantities of heme-iron proteins, iron-sulfur proteins, complex iron-sulfur proteins, as well as in phenylalanine hydroxylase, an enzyme with iron in an undetermined coordination.     

A chemiluminescence automatic analyser for the measurement of biological compounds

A compact automated analyser which could analyse constituents in biological fluids with a small sample volume and in a short time has been developed. The instrument was composed of a flow injection analysis system equipped with chemiluminometric detection and an immobilized enzyme column reactor used in combination. Chemiluminescence has high sensitivity, and its reaction proceeds very quickly. Furthermore, an immobilized enzyme column reactor can produce a sufficient amount of hydrogen peroxide from compounds in serum in a short time. When enzymes are used as reagents for the analysis of substances in blood or blood serum, the final signals emitted by different enzyme reactions are usually not only hydrogen peroxide but also ammonia, NAD(P)H and so on. However, the practical chemiluminescence method for ammonia and NAD(P)H has not been established. We have discovered a new practical method for ammonia and NAD(P)H using an enzyme column reactor consisting of both immobilized L -glutamate dehydrogenase and L -glutamate oxidase. The determinations of glucose and uric acid in serum by chemiluminometry after production of hydrogen peroxide by the respective oxidases are presented. A newly chemiluminometric determination of ammonia, NAD(P)H and its applications to other enzymatic analyses that give ammonia and NAD(P)H as a final signal are also described.     

ISFET based enzyme sensors

This paper reviews the results that have been reported on ISFET based enzyme sensors. The most important improvement that results from the application of ISFETs instead of glass membrane electrodes is in the method of fabrication. Problems with regard to the pH dependence of the response and the dynamic range as well as the influence of the sample buffer capacity have not been solved. As a possible solution we introduce a coulometric system that compensates for the analyte buffer capacity. If the pH in the immobilized enzyme layer is thus controlled, the resulting pH-static enzyme sensor has an output that is independent of the sample pH and buffer capacity and has an expanded linear range.     

Studies on the kinetics of immobilized enzyme using a recycling enzyme reactor system

A method of measuring kinetic parameters of immobilized enzyme with a recycling enzyme reactor system is described. By analyzing the plot of the dimensionless variable Ln(1-X)/X versus the time needed for a unit conversion, t/X, the mechanism of enzymatic reaction can be recognized and then its basic parameters can be evaluated. On the basis of the experimental data measured by P.R. Coulet et al., it has been proposed that the successive degradation of maltodextrins by the collagen membrane-bound amyloglucosidase was a product glucose inhibition reaction and their corresponding constants have been found with this method.     

Isolation and characterization of the N-domain of bovine angiotensin-converting enzyme

A method for preparation of a catalytically active fragment of bovine lung angiotensin-converting enzyme (ACE) has been developed. It includes limited proteolysis of the full-length somatic form of the enzyme by trypsin. The resulting fragment corresponds to the N-terminal domain of angiotensin-converting enzyme. The influence of chloride and sulfate anions on the enzymatic activity of this fragment has been investigated, and kinetic parameters for hydrolysis of synthetic tripeptide substrates catalyzed by the N-domain of ACE have been determined. Comparison of these parameters with those obtained for full-length somatic bovine ACE suggests that in the bovine somatic ACE molecule active centers located in various domains may function interdependently.     

Inhibition by substrate of fructose 1,6-bisphosphatase purified from rat kidney cortex. Calculation of the kinetic constants of the enzyme

Fructose 1,6-bisphosphatase is a typical enzyme that is severely inhibited by its own substrate. This makes it difficult to determine all the parameters involved in its kinetics. It has been shown recently that if Vm is satisfactorily estimated the remaining parameters can be determined using the Hill plot (Bounias, M. (1988) Biochem. Int. 17, 147-154). The enzyme has been purified from rat kidney cortex nearly to homogeneity, and its kinetic constants have been calculated using a rigorous algebraic method. The most interesting result is that the substrate is unable to bind to the free enzyme as an inhibitor, which indicates that the enzyme lacks an allosteric site for hexose bisphosphates.     

One-step enzyme immunochromatographic assay for theophylline

The convenience of the previously described enzyme immunochromatography method for visually quantifying theophylline in whole blood has been improved with the development of a one-step protocol. The capillary migration and color generation in the two-step enzyme immunochromatographic assay have been combined into a single step. Ascorbic acid is used as a signal inhibitor to delay enzymatic color product formation until the inhibitor itself is consumed. The concept of internal delay reaction is presented and the mechanism of ascorbate's action as an inhibitor to temporarily delay color generation is described. The internal delay reaction has been applied to a practical one-step quantitative visual enzyme immunochromatographic assay for theophylline in whole blood.     

Direct measurement of enzyme activity with infrared spectroscopy

A direct approach to enzyme activity measurements is presented. Vibrational spectroscopy can monitor the progress of enzymatic reactions because the vibrational spectrum of substrates and products usually differs. This is demonstrated by the example of ATP hydrolysis by Ca(2+)-ATPase: The substrate concentration can be followed using the infrared absorption of the alpha- and beta-PO(2)(-) phosphate groups of ATP, and the product concentration can be followed using the PO(3)(2-) absorption of P(i) and of the beta-phosphate of ADP. The results of the infrared spectroscopic measurement of ATPase activity and of an independent activity assay agree very well. The main advantage of the infrared method is that it observes the reaction of interest directly--that is, no activity assay that converts the progress of the reaction into an observable quantity is required.     

Influence of polymerization of D-amino acid oxidase on the behavior of the enzyme immobilized on chitosan by covalent fixation

D-Amino-acid oxidase is a flavoprotein using FAD as cofactor. The enzyme has been immobilized in the presence of FAD on a non-porous matrix: chitosan. This support is covalently bound to the enzyme with glutaraldehyde as cross-linking reagent. It is characterized by a good mechanical resistance to mechanical stirring. The enzymatic assays have been performed in batch reactor with D-phenylglycine as substrate by a spectrophotometric method which is based on the variation of the absorbance at 252 or 280 nm. The behaviour of the biocatalysts has been studied during repeated assays of 1 h at 25 degrees C in the absence of exogenous FAD. The experimental results have been compared with those obtained with the soluble enzyme tested in the presence or in the absence of FAD. The dependence of D-amino-acid oxidase on FAD concentration has been studied. Immobilized enzyme on chitosan appears to be less sensitive to the association-dissociation equilibrium of FAD. This property and the capacity of the enzyme to polymerize spontaneously in solution according to the experimental conditions have been established. The fact that the enzyme can exist in various oligomeric forms is of major importance because its catalytic expression is dependent of this phenomenon. The polymerization is known to be responsible for a decrease of the maximal rate V of the enzyme. It has also been shown that in the same way this decrease was accompanied by an improvement of the affinity of enzyme for substrates. Furthermore, the value of the dissociation constant of the apoenzyme-FAD complex is significantly smaller as the degree of polymerization is high. The conclusion is that the dissociation of the cofactor can be avoided if the immobilization step is carried out at high concentration of enzyme which is favourable to its polymerization.     

Transient-phase kinetics of enzyme inactivation induced by suicide substrates

This paper deals with the kinetic study of reaction mechanisms with enzyme inactivation induced by a suicide substrate in the presence or absence of an auxiliary substrate and in conditions of excess of substrates in relation to the enzyme concentration and vice versa. A transient-phase approach has been developed that enables explicit equations with one or two significant exponentials to be obtained, thereby showing the dependence of product concentration on time. The validity of these equations has been checked, and a comparison made with those previously obtained by other authors. We propose an experimental design to determine the corresponding parameters and kinetic constants. The simplicity of our method allows a systematic application to more complex mechanisms.     

A simple method for measurement of phosphoramidon-sensitive endothelin converting enzyme activity.

We have developed a rapid and convenient assay for measurement of the action of endothelin (ET) converting enzyme (ECE) using the scintillation proximity assay (SPA) principle. On incubation of [125I]big ET-1 at 37 degrees C for 0.5-6 hr with an enzyme preparation, the reaction was terminated by the addition of an ET-1-specific antibody formulated in a buffer designed to shift the pH to alkaline. The antibody was allowed to come to equilibrium for 1 hr at room temperature and the amount of ET-1 produced, detected in a single step by the addition of protein A SPA beads. Using this assay, ECE activities of enzyme preparations obtained from porcine cultured endothelial cells and rat lung were clearly detected. These activities were inhibited by phosphoramidon in a concentration-dependent manner. The SPA based assay is homogeneous requiring no separation steps and takes a half day to complete. This method is therefore suitable for the high throughput screening of potential ECE inhibitors.     

Half-time analysis of the kinetics of irreversible enzyme inhibition by an unstable site-specific reagent

The half-time method for the determination of Michaelis parameters from enzyme progress-curve data (Wharton, C.W. and Szawelski, R.J. (1982) Biochem. J. 203, 351-360) has been adapted for analysis of the kinetics of irreversible enzyme inhibition by an unstable site-specific inhibitor. The method is applicable to a model in which a product (R) of the decomposition of the site-specific reagent, retaining the chemical moiety responsible for inhibitor specificity, binds reversibly to the enzyme with dissociation constant Kr: (formula; see text). Half-time plots of simulated enzyme inactivation time-course data are shown to be unbiased, and excellent estimates of the apparent second-order rate constant for inactivation (k +2/Ki) and Kr can be obtained from a series of experiments with varying initial concentrations of inhibitor. Reliable estimates of k +2 and Ki individually are dependent upon the relative magnitudes of the kinetic parameters describing inactivation. The special case, Kr = Ki, is considered in some detail, and the integrated rate equation describing enzyme inactivation shown to be analogous to that for a simple bimolecular reaction between enzyme and an unstable irreversible inhibitor without the formation of a reversible enzyme-inhibitor complex. The half-time method can be directly extended to the kinetics of enzyme inactivation by an unstable mechanism-based (suicide) inhibitor, provided that the inhibitor is not also a substrate for the enzyme.     

Methods for the measurement of a bacterial enzyme activity in cell lysates and extracts

The kinetic characteristics and regulation of aspartate carbamoyltransferase activity were studied in lysates and cell extracts of Helicobacter pylori by three different methods. Nuclear magnetic resonance spectroscopy, radioactive tracer analysis, and spectrophotometry were employed in conjunction to identify the properties of the enzyme activity and to validate the results obtained with each assay. NMR spectroscopy was the most direct method to provide proof of ACTase activity; radioactive tracer analysis was the most sensitive technique and a microtitre-based colorimetric assay was the most cost-and time-efficient for large scale analyses. Freeze-thawing was adopted as the preferred method for cell lysis in studying enzyme activity in situ. This study showed the benefits of employing several different complementary methods to investigate bacterial enzyme activity.     

An artifact in measurement of S phase initiation and its implication for the kinetics of S phase-specific enzyme activities

It is shown that the different onset of S phase as measured by autoradiography vs cumulative thymidine uptake is an artifact. We consequently propose that S phase-specific enzyme activities may accumulate a few hours prior to the actual initiation of DNA synthesis. A “pre-S” DNA synthesis that can be readily detected only by autoradiography has been proposed. Published data show that DNA synthesis in cultured animal cells is initiated approx. 2 h later when measured by cumulative incorporation of [³H]thymidine ([³H]TdR) as compared with autoradiography. We show here that the difference is in reality an artifact, owing to not taking into account both gradual, asynchronous entry of cells into S phase, as well as time-dependent accumulation of radioactivity into each cell after it has entered S phase. Combination of these two factors leads to the conclusion that [³H]TdR should be incorporated approximately as the square of time following entry of the first cell into S. Taking this into account, the two methods then are in agreement, as predicted. This argument also applies to the enzyme activities shown to increase with DNA synthesis in synchronized cultures. Such an enzyme accumulation really could begin some time earlier than indicated by conventional plots of cumulative enzyme activity vs time and may, in fact, precede the onset of S by a few hours.