Arne Lundin

Continuous monitoring of ATP-converting reactions by purified firefly luciferase

The time course of the bioluminescence obtained with a partially purified firefly luciferase preparation has been studied. At ATP levels less than 10−6m the light emission could be maintained essentially constant for several minutes, if the luciferase was not subjected to product inhibition or other inactivating processes. This could be achieved by performing the reaction at appropriate pH and concentration of luciferin and luciferase. Under these conditions continuous measurement of light emission may be used for nondestructive monitoring of ATP-converting reactions, since the emission will be proportional to the ATP concentration in each instant. The continuous monitoring of ATP concentration by firefly luciferase was used for kinetic determination of enzymes and metabolites and for endpoint analysis of metabolites. It was found to be extremely sensitive and convenlent for routine applications.

Analytical information obtainable by evaluation of the time course of firefly bioluminescence in the assay of ATP.

Abstract The analytical accuracy of the firefly assay of ATP has been studied and is discussed with special reference to interference with the time course of the bioluminescence. Influence from rate of mixing and from interfering substances is described. Methods for quantitation of the bioluminescence using different phases of the time course have been compared with respect to susceptibility to analytical interference. In most cases the method least susceptible to interference was measurements of the rate of the initial rise of the bioluminescence. Purified as well as commercial luciferase reagents have been used, and analytical characteristics are compared. Correction for analytical interference by standard addition techniques was shown to result in erroneous assay in a bacterial extract.

Use of firefly luciferase in ATP-related assays of biomass, enzymes, and metabolites.

The kinetics of ATP reagents not affected by product inhibition or other forms of inactivation of luciferase during the measurement time has been clarified. Under these conditions the decay rate of the light emission expressed as percentage per minute is a measure of luciferase activity and can be given as the rate constant k (min-1), directly reflecting the degradation of ATP in the luciferase reaction. Three types of reagents with different analytical characteristics and different application possibilities have been identified. Stable light-emitting reagents are suitable for measurements of ATP down to 1000 amol. This is the only type of reagent suitable for monitoring ATP-converting reactions, i.e., assays of enzymes or metabolites, assays of oxidative phosphorylation, photophosphorylation, and so on. A higher luciferase activity resulting in a slow decay of the light emission by approximately 10% per minute (k = 0.1 min-1) gives a reagent suitable for measurements down to 10-100 amol. The slow decay of light emission allows use of manual luminometers without reagent dispensers. A further increase of the luciferase activity resulting in a decay rate of approximately 235% per min (k = 2.35 min-1) and only 10% of the light emission remaining after 1 min is suitable for measurements down to 1 amol corresponding to half a bacterial cell. With this type of flash reagent the total light emission can be calculated from two measurements of the light intensity on the decay part of the light emission curve. This new measure is not affected by moderate variations in luciferase activity, but only by changes in quantum yield and self-absorption of the light in the sample. Flash-type reagents require the use of reagent dispensers. The stringent requirements for ATP-free cuvettes, pipette tips, and contamination-free laboratory techniques make it unlikely that flash reagents would be useful in nonlaboratory surroundings. A potential application for this type of reagent is sterility testing. In general, it is concluded that one should select the ideal ATP reagent carefully for each application. Obviously the reagents used in a particular application do not have to match the decay rates given earlier exactly. However, various applications of the ATP technology and the properties of manual and automatic luminometers fall quite nicely into categories corresponding to the properties of the three reagents described. The rapidly growing interest in ATP technology has already resulted in the development of a greater variety of luminometers, from hand-held instruments to high-throughput systems. The continuation of efforts in both reagent and instrument development will undoubtedly result in many new applications.

[4] Estimation of biomass in growing cell lines by adenosine triphosphate assay

Publisher Summary This chapter describes the steps involved in estimation of biomass in growing cell lines by luminometric ATP assay. Methods for studying the reliability of the results are described. In particular, estimation of energy charge (EC) by a new method for assay of ATP, ADP, and AMP was found to be valuable. The activity of several adenosine nucleotide converting enzymes as a function of the energy charge—that is, (ATP + 0.5 ADP) / (ATP + ADP + AMP)—indicates that the energy charge in intact metabolizing cells should be stabilized in the interval 0.8–0.9. By measuring all three adenine nucleotides, the measure of the physiological status of the cells is obtained that provides information on whether ATP can be used to estimate biomass or if the energy metabolism is disturbed. By the method described in the chapter, all three adenine nucleotides are determined in a single aliquot from the cell extract. Measurement of energy charge gives a reliable estimation of the accuracy of using ATP as a biomass parameter. Growth monitoring by assay of adenine nucleotides includes the following steps: (1) cultivation of cells, (2) sampling and pretreatment of samples, (3) extraction of adenine nucleotides from cells, (4) luminometric assay of adenine nucleotides, and (5) correlation to other biomass estimations.

Automatic luminometric kinetic assay of glycerol for lipolysis studies.

A kinetic assay for glycerol based on the glycerokinase and firefly luciferin-luciferase reactions was developed for the purpose of allowing automatic analysis of large series of incubates from human fat cells. The method includes simplified sampled pretreatment and fully automated analysis, recording, and calculation of data. The upper limit of the dynamic range of the assay was extended from a glycerol concentration in cuvette of 2 up to 100 microM. This is due to a principally new method to linearize the nonlinear part of the standard curve. Recovery experiments reveal 100% glycerol recovery. Within-run and between-run precisions are high with coefficients of variation of between 1.2 and 3.7%. Samples are stable for up to 6 months in an ordinary freezer. The detection limit expressed as 2 SD of the blank result is 0.07 microM glycerol in cuvette, which is slightly lower when compared to the previous luminometric methods. The apparent blank, which is typically 0.4-1.0 microM glycerol in cuvette, has shown significant contributions only from bovine serum albumin, 0.03 microM, and glycerokinase, 0.01 microM. The analytical performance and degree of automation of the glycerol method described in this paper makes it well suited for serial studies of lipolysis in human fat cells in the presence of lipolytic or antilipolytic agents.

A new linear plot for standard curves in kinetic substrate assays extended above the Michaelis-Menten constant: Application to a luminometric assay of glycerol

In conventional kinetic substrate assays the standard curve is plotted as observed reaction rate, upsilon obs, versus added substrate concentration, Sadd, and has a linearity limited to Sadd much less than Km. From this plot the blank reaction rate, upsilon bl, is easily estimated but not the contaminating substrate concentration, Scon, present in reagents (unless it is the only blank source). Thus the actual substrate concentration, S = Scon + Sadd, cannot be estimated as required for the various linear plots based on the Michaelis-Menten equation. We have derived an expression, (upsilon obs - upsilon bl)/Vapp = Sadd/(Kmapp + Sadd), containing only those parameters measured for a conventional standard curve (Vapp and Kmapp are obtained from a plot of (upsilon obs - upsilon bl) versus (upsilon obs - upsilon bl)/Sadd). A plot of (upsilon obs - upsilon bl)/Vapp versus Sadd/(Kmapp + Sadd) can be used as a standard curve with the following advantages over the conventional standard curve: (a) For all kinetic substrate assays it is identical and connects the points (0, 0) and (1, 1). Thus deviations from true Michaelis-Menten kinetics or erroneous kinetic constants are easily detected. (b) Since it is linear even above Km, the analytically useful range is considerably extended. (c) For assays with a wide dynamic range it can be used in lin-lin or log-log form. The procedure is illustrated for a kinetic assay of glycerol (Kmapp = 40 mumol/liter). The plot was found to be entirely linear in the range 0.07-100 mumol/liter (glycerol concentration in cuvette).

Glucose determination in samples taken by microdialysis by peroxidase-catalyzed luminol chemiluminescence

An automatic, luminometric assay of glucose in samples of the extracellular water space obtained by microdialysis is described. The assay involves oxidation by glucose oxidase (EC 1.1.3.4) and mutarotation of glucose by aldose mutarotase (EC 5.1.3.3.). The H2O2 formed is subsequently determined in a reaction catalyzed by horseradish peroxidase (EC 1.11.1.7) using luminol as electron donor. The assay is linear between 0.01 and 1 nmol in the cuvette. The detection limit, defined as 3 standard deviations of the reagent blank, was 0.008 mumol/liter in the cuvette. A complete oxidation of glucose is obtained within 4 min and 25 samples are automatically assayed within 75 min. Addition of microdialysate sample obtained from human adipose tissue in vivo did not interfere with the standard curves. Glucose added to microdialysate resulted in a complete recovery compared to a H2O2 standard. Analytical interference from different factors was investigated. No interference was observed up to the following concentrations: 5 mumol/liter epinephrine, 1 mumol/liter norepinephrine, 100 mumol/liter insulin, 500 mumol/liter pyruvate, 50 mmol/liter lactate, and 1 mumol/liter ascorbate. The glucose values with the present method correlated strongly (r = 0.984) with values obtained using a routine method involving glucose oxidase and peroxidase.

Evaluation of the adenosine triphosphate test in the diagnosis of urinary tract infection

Determination by bioluminescence of the bacterial adenosine triphosphate (ATP) level in urine was evaluated as a method for detection of bacteriuria in 1126 women with symptoms of UTI and 530 attending for follow-up. Conventional urine culture was used as reference method. The criterion for bacteriuria was growth of ≥ 105 cfu/ml, giving a prevalence of 0.60. ATP levels of <10 nmol/l and >50 nmol/l indicated abacteriuria and bacteriuria, respectively, whereas intermediate concentrations required culture if the nitrite test was negative. With this diagnostic strategy the sensitivity and specificity was 0.96 and 0.90 at the first visit and 0.90 and 0.98 at follow-up. With some methodological improvement the ATP test could be useful in medium-sized and small laboratories.

Female Urinary Tract Infection in Primary Health Care: Bacteriological and Clinical Characteristics

Female patients with symptoms of urinary tract infection (n = 1136) were studied in primary health care with respect to (a) clinical symptoms as predictors of bacteriuria; (b) relation between aetiological agent and clinical picture, especially for P-fimbriated Escherichia coli; and (c) clinical findings in cases with 10(2)- less than 10(5) CFU/ml of E. coli. Prevalence of bacteriuria (greater than or equal to 10(5) CFU/ml) was 61%. Concurrence of urgency/frequency and dysuria, short duration of symptoms and hematuria increased the probability of bacteriuria and were also significantly more frequent among cases with low counts of E. coli (10(2) less than 10(5) CFU/ml in pure culture or mixed flora) than among cases with sterile urine, indicating an aetiological role of E. coli in many of those cases. Infections with P-fimbriated E. coli were as benign as the P-fimbriae-negative. The rate of P-fimbriation was 29% in specimens containing greater than or equal to 10(5) CFU/ml of E. coli, 30% among specimens with less than 10(5) CFU/ml in pure culture and 10% in specimens containing less than 10(5) CFU/ml of E. coli in mixed culture. Patients infected with Klebsiella, Enterobacter or Proteus did not show a higher rate of previous urinary tract disease or anomalies.

[15] Flow-injection analysis with immobilized chemiluminescent and bioluminescent columns

Publisher Summary Flow analysis using immobilized chemi- and bioluminescent packed-bed enzyme reactors as detectors is rapid, sensitive, and precise. The reusability and stability of the immobilized enzyme columns make them potential and low-cost tools for automated determinations of different analytes in both research and routine analysis. There are many matrices and many different procedures used for enzyme immobilization. High coupling efficiency, high remaining activity, good stability of the final product during storage and continuous use, and good mechanical stability of the support are the main criteria when the immobilization procedure is optimized. For chemi- and bioluminescent systems there is also a special demand for the matrix and the coupling reaction: the enzyme–matrix conjugate should not absorb any light at 400–600 nm. This chapter describes the immobilization of peroxidase, choline oxidase, firefly luciferase, and the bacterial bioluminescence enzymes with different dehydrogenases, including the use of resulting enzyme preparations in flow-injection analysis of some metabolites.