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J Biomol Tech. 2007 September; 18(4): 185–187.
PMCID: PMC2062561

Rapid Kinetic Microassay for Catalase Activity

Abstract

Catalase is a commonly assayed enzyme found in many bacteria and eukaryotes. In this report, we examined the applicability of a kinetic microassay to quantify catalase from two different sources. The assay was found to be linear over a wide range (0.1–1.0 units), but was limited at high values (>1 unit) by oxygen evolution. Nonetheless, the micorassay allows simultaneous evaluation of many samples (up to 96) in a short time (<5 min) and is thus well-suited to applications, such as high-throughput screening, where many parallel assays are required.

Keywords: catalase, spectrophotometric assay, high-throughput screening, microplate, hydrogen peroxide

Catalase is a well-studied enzyme produced by a wide spectrum of eukaryotic and prokaryotic organisms.1 The enzyme is generally quantified using assays that are based on either the decrease in absorbance of hydrogen peroxide at λ = 240 nm2 or by measuring oxygen release with Clark-type electrodes.3 As kinetic assays, these are usually performed in single-cell analytical instruments. The lack of a validated microassay is thus limiting, even though it is often desirable to test large numbers of samples in parallel, in order to examine the synthesis of enzymes in response to specific inducers or in mutants that are deficient in the synthesis of one or more of the enzymes. The availability of microtiter plate readers with extended UV capability, the manufacture of microtiter plates with polymers having extended UV transparency, and the need to assay small amounts of enzyme activity prompted us to examine the possibility of developing a broadly applicable catalase microassay. Though microtiter plate assays have been used to measure catalase activity,4 there has not been a focused technical study to validate the methodology. In the present study, we tested a modification of the Beers and Sizer assay2 that can be used to conveniently assay large numbers of samples in parallel and evaluate the applicability of this microassay based on its sensitivity, assay range, and reproducibility.

MATERIALS AND METHODS

Sources of Catalase

Bovine liver catalase (Sigma C-30) was used as a quality control to evaluate the sensitivity and accuracy of this method. Escherichia coli K-12 strain and catalase deletion derivatives were created using a chromosomal deletion method (Table 1).5

TABLE 1
E. coli Strains Used in This Study

Preparation of E. coli cell-Free Extract

The crude cell extracts were prepared as described previously.6 Briefly, cells from overnight cultures were washed and resuspended in 0.05 M phosphate buffer (pH 7.0). The cells were lysed by sonication in an inverted-cup sonicator at 4°C with pulse-on time 15 sec for every 30 sec. The cell lysates were collected after centrifugation at 12,000 g for 20 min at 4°C. The protein concentration in the cell lysates was determined using the standard Bradford colorimetric assay, with bovine serum albumen as the standard.7

Catalase Microassay

Bovine liver catalase (Sigma C-30) was diluted with 0.05 M phosphate buffer (pH 7.0). Varying quantities of catalase, ranging from 0.01 to 2.00 units, were added to a 96-well flat-bottom UV-transparent microtiter plate (Thermo Scientific, Ottawa Ontario). Hydrogen peroxide was diluted in 0.05 M phosphate buffer (pH 7.0) to a final concentration of 5 mM. A volume of 250 μL of substrate solution was added to each well of the microtiter plate rapidly, using a repeating pipette. The plate was then immediately scanned in a spectrophotometer (Multiskan Spectrum, Thermo Scientific, Ottawa ON) at λ = 240 nm every 10 sec for 5 min at 22°C. Cell-free extracts from various E. coli strains were prepared and scanned in the same manner. Catalase activity was calculated based on the rate of decomposition of hydrogen peroxide, which is proportional to the reduction of the absorbance at λ = 240 nm. The catalase activities of E. coli extracts were normalized to total cellular protein in the lysate and are expressed as units per mg of protein.

RESULTS AND DISCUSSION

In the present study, we report a rapid and sensitive modification of the standard kinetic spectrophotometric assay for catalase activity.2 Using this method, up to 96 reactions can be performed simultaneously in a 96-well UV-transparent microtiter plate within 5 min. We evaluated the sensitivity and accuracy of this method using bovine catalase of known concentrations as a proficiency control. The rate of decomposition of H2O2 was linear throughout the reaction, with catalase levels ranging from 0.05 to 1.0 units in a total reaction volume of 250 μL (Figure 1). We found that catalase, at quantities above 2 units, resulted in excessive oxygen formation and consequent bubbling, hence limiting the assay. The lower limit of the assay was found to be 0.05 units, making the assay comparable in sensitivity to the standard Beers and Sizer assay.2 Therefore, the optimal range of this assay is between 0.05 units and 1.0 unit in a reaction volume of 250 μL. The catalase activity of replicate samples was calculated based on the rate of decrease in absorbance at λ = 240 nm using the molar extinction coefficient of hydrogen peroxide, and corrected for pathlength.2 To assess the accuracy of the assay method, the calculated activities were compared with the actual activities of the catalase standards. The response curve between calculated activities using this method and the actual activities was linear with a slope of 0.95, indicating the microtiter method provides an accurate estimate of activity within the defined assay range (Figure 2).

FIGURE 1
Decomposition of hydrogen peroxide over time by varying quantities of catalase. The change in concentration of hydrogen peroxide is proportional to the decrease in absorbance at λ = 240 nm. The rate of reaction, as measured by the slope of the ...
FIGURE 2
Correlation between the rate of decrease in absorbance of hydrogen peroxide and catalase activity. The rate of decrease in absorbance at λ = 240 nm correlates with the levels of catalase activity in a linear relationship with a correlation coefficient ...

To evaluate the applicability of the microtiter assay to other types of samples, we measured the catalase activities of four E. coli strains harboring deletions of catalase-encoding genes, including wild-type and katE and/ or katG null mutants (Table 1). Cell-free extracts prepared by sonication were diluted to avoid bubble formation during the assay. The catalase activities of the E. coli cell extracts were calculated from the rate of decrease in absorbance using the standard ΔA240 vs. catalase activity curve established previously (Figure 2). As expected, the wild-type strain exhibited the highest levels of catalase activity, as it possesses both functional catalase/ peroxidase–encoding genes, katE and katG, respectively. The ΔkatE and ΔkatG strains exhibited reduced activities. Lacking both catalase-encoding genes, the ΔkatEΔkatG strain had no detectable activity (Figure 3). The consistency of the results obtained using the present method and the conventional single-cuvette method (data not shown) using the Beers and Sizer assay2 suggests that this method can be readily applied in kinetic assay of catalase regardless of the enzyme source.

FIGURE 3
Catalase activities of E. coli strains harboring various catalase deletions. The highest catalase activity was detected in wild type K-12 E. coli strain containing both catalase and catalase/peroxidase encoding genes. The ΔkatE and ΔkatG ...

In comparison to the conventional catalase assay using individual cuvettes, the microassay method reported in this study substantially decreases the assay time required and facilitates the simultaneous determination of a large number of samples. Employing bovine catalase as a control, we have shown that this microassay method is both accurate and sensitive. Its broad applicability was demonstrated by the quantification of catalases from bacterial samples. The parallel use of a standard curve using catalase of known concentrations further simplifies data analysis. The quantity of catalase was identified as a critical factor for valid application of the assay. Due to the small reaction volume (250 μL) and short path length (0.5 cm), the formation of air bubbles caused by vigorous reactions may result in nonlinearity. We have identified the optimal assay range of catalase to be between 0.05 and 1.0 units.

ACKNOWLEDGMENTS

We thank Alison Fine for proofreading the manuscript and the funding agencies of the HES lab, including the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR).

REFERENCES

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