Assayofsuperoxidedismutaseactivity1

Assay of superoxide dismutase activity by combining electrophoresis and densitometry

Abstract. A modified technique was developed to assay superoxide dismutase (SOD) activity by combining polyacrylamide gel electrophoresis and densitometry. After electrophoresis on native polyacrylamide gels, the negative banding corresponding to the SOD activity was visualized by soaking the gels in nitroblue tetrazolium then riboflavin, and finally exposing to light. Effects of the banding of SOD activity induced by different soaking durations and light intensities were evaluated in this system. The optimal soaking duration was determined to be 15 min for each of the two soaking steps, while the optimal exposure was 30 mEm-2s-1 for 15 min. The gels were then immediately scanned with a laser densitometer, and the readings of the samples corresponding to their total SOD activity were obtained by processing the image. A standard curve was prepared with a serial dilution of partially purified SOD, whose activity was previously determined by using a spectrophotometric method. The total SOD activity of an unknown sample could be obtained by interpolating its reading to the standard curve. The activity of a single SOD isozyme of a sample could also be obtained with the same procedure. The technique was ten times more efficient than the spectrophotometric method. The interference coming from non-SOD substances in the crude extract could be removed by electrophoresis. The standard deviations of the SOD activity of the crude extracts from rice seedlings, papaya, and tobacco leaves measured with the technique were less than 9%, 7%, and 8% (for each n = 6, on 6 gels), respectively.

Keywords: Densitometry; SOD isozyme; Superoxide dismutase.

Abbreviations: DETAPAC, diethylenetriamine pentaacetic acid; ED, electrophoretic-densitometry; NBT, nitroblue tetrazolium; SOD, superoxide dismutase; TEMED, tetramethylenediamine.

Introduction

Superoxide radical (•O2_) is generated as a by-product in aerobic organisms from a number of physiological reactions such as the electron flow in the chloroplasts and mitochondria and from some redox reactions in cells. It can react with hydrogen peroxide (H2O2) to produce hydroxyl radical (•OH_), one of the most reactive molecules in the living cells. Hydroxyl radical can cause the peroxidation of membrane lipids, breakage of DNA strands, and inactivation of enzymes in cells (for reviews, see Bowler et al., 1992; Mehdy, 1994). To ameliorate the damage caused by hydroxyl radical formed from superoxide radical and hydrogen peroxide, organisms have evolved mechanisms to control the concentration of the two reactants. Superoxide dismutase (SOD, EC 1.15.1.1) is a group of isozymes functioning as superoxide radical scavenger in the living organisms. The reaction of SOD is as follows: 2H+ + 2 •O2_ ® H2O2 + O2

the produced hydrogen peroxide is then detoxified by catalase or peroxidase.

The expression of SOD genes is regulated both spatially and developmentally at least in maize (Zhu and Scandalios, 1993) and rice (unpublished data). SOD activity is also induced by diverse stresses (Bowler et al., 1992), presumably because of the increase in the concentration of superoxide radical in cells under those conditions. Obviously, SOD is an important enzyme family in living cells for maintaining normal physiological conditions and coping with stress. However, the study of SOD gene expression regulation at the end product level has been handicapped by the lack of a convenient method for quantifying the activity assay of the isozymes. Most of the work concerning SOD gene expression regulation has been based on RNA gel blot analysis. Little is known about the activity change of the SOD isozymes of plants in the developmental course and in response to stresses. SOD activity is commonly assayed spectrophotometrically, e.g., the method first defined by McCord and Fridovich (1969) and modified by Oberley and Spitz (1985). But it is both labor-intensive and time-consuming. We have developed a convenient technique for assaying the SOD activity by combining electrophoresis and densitometry and have called it the ED scheme. This technique is based on Beauchamp and Fridovich's method (1971), but is quantitative rather than qualitative. The ED scheme is more than ten times more efficient than the spectrophotometric method. The SOD activity of unknown samples can be derived from comparing with a SOD standard whose activity was measured by the spectrophotometric method. In addition to its efficiency, the technique can assay the activity of a single SOD isoform on the polyacrylamide gel and exclude the interference coming from non-SOD molecules in the tissue crude extract, which is not possible using the spectrophotometric method.

Materials and Methods

Nitroblue tetrazolium (NBT), diethylenetriamine pentaacetic acid (DETAPAC), xanthine and xanthine oxidase were purchased from Sigma. Riboflavin and tetramethylenediamine (TEMED) were purchased from Serva Chemical Company.

For the preparation of samples for the assay of SOD activity, 3 g of rice seedlings, tobacco and papaya young leaves were harvested and frozen with liquid nitrogen, ground into a fine powder, and then mixed with 3 ml of extraction buffer (0.15 M Tris, pH 7.5). The samples were centrifuged at 4°C, 14 k × g for 10 min. Centrifugation was repeated 2_3 times to clear all the debris. The supernatant was then transferred into microtubes, stored at -20 °C, and centrifuged again before use. SOD standard was a partially purified mungbean SOD obtained from the King Car Food Industrial Cooperation.

Electrophoresis was carried out at 4°C according to a modified procedure of Gabriel (1971) with 1.5 mm of 10% polyacrylamide mini-slab gel in standard tris-glycine buffer (pH 8.3). Samples were loaded into each well and then electrophoresed at 80 V through the stacking gel for 15 min and 120 V through the separating gel for 60 min. After electrophoresis, a modified photochemical method of Beauchamp and Fridovich (1971) was used to locate SOD activities on gels. The gel was first soaked in 25 ml of 1.23 mM NBT for 15 min, briefly washed, then soaked in the dark in 30 ml of 100 mM potassium phosphate buffer (pH 7.0) containing 28 mM TEMED and 2.8 × 10-2 mM riboflavin for another 15 min. The gel was briefly washed again, and then illuminated on a light box with a light intensity of 30 mEm-2s-1 (measured by LI-COR LI 1000) for 15 min to initiate the photochemical reaction. All the procedures were carried out at room temperature, and the two soaking steps were shaken at 75 rpm.

The gel was scanned with a laser densitometer (Molecular Dynamics) immediately after the photochemical reaction. To measure the total SOD activity, each sample lane was individually framed with a suitable size rectangle when processing the image on the computer, ensuring that every SOD band of a lane fell into the rectangle. In addition to the sample rectangles, a number of blank rectangles were framed on the same gel for calibrating the background of the sample rectangles. The reading corresponding to the SOD activity in one sample rectangle could be calculated according to the formula: Reading of SOD activity = (average pixel reading of the blank rectangles) × (the pixel number of the sample rectangle) _ (the total reading of the sample rectangle). The outcome represents a different degree of staining resulting from the total SOD activity of each sample. A single SOD isoform can be processed in a similar way by framing the location of the desired isoform as the sample rectangle.

Spectrophotometric assay of SOD activity was carried out by adapting the procedure of Oberley and Spitz (1985). For a 20-cuvette assay, the following reagents were added to a test tube: 13.8 ml of 50 mM potassium phosphate buffer (pH 7.8) containing 1.33 mM DETAPAC; 0.5 ml of 2.45 mM NBT; 1.7 ml of 1.8 mM xanthine. The total volume was 16 ml, enough to dispense 0.8 ml into each of the 20 cuvettes.