Botanical Bulletin of Academia Sinica, Vol. 37, 1996

The method using NBT as a superoxide radical competitor and a color indicator was also explored to qualitatively locate SOD on polyacrylamide gels at the time when cytochrome c was replaced by NBT. In the NBT negative staining system, after the gels have been soaked with NBT then riboflavin, exposing them to light causes the riboflavin to generate a superoxide radical flux in the presence of oxygen and TEMED. NBT and SOD in the gels compete for the superoxide radical at the same time. At the locations where SOD exists, the gel remains transparent, in contrast to those areas without SOD where the gel becomes purple-blue due to reduced NBT. Although the staining system was set up more than twenty years ago, it has not been modified and optimized to quantify SOD activity. Our experience and some reports showed the potential to modify the qualitative SOD staining system to quantify the activity of SOD. The isozymes of the SOD family can be separated by electrophoresis, their banding intensities are proportional to the loaded amount of SOD samples in fixed conditions, and the developed gels can be easily scanned with a laser densitometer. These three factors provided the basis for assaying the activity of the total or a single SOD isozyme on a gel with the ED scheme. Further more, in the mini-slab-gel system, to run four gels which have ten wells each at the same time is easy. This provided the efficiency for the technique. Loading a suitable quantity of sample in each well is a crucial first step in the ED scheme. If the sample is overloaded, some SOD isozyme with higher activity will be under-estimated due to the saturation effect. Thus, the loaded sample amount should fall into the linear range of both the standard curve and the sample curve. Because the curves of crude extracts showed a wider linear range than that of purified SOD due to the presence of more isozymes, readings of samples falling in the linear range of the standard curve prepared with a purified SOD are considered to be safe. However, the variation of the readings was also quite high when the loaded amount was low. In this work, the suitable range for the interpolation was between 300 and 1,300.

The intensity of SOD bands was affected by the 2-step soaking and illumination procedure. The longer the soak durations, the darker and blurrier the SOD bands. Some SOD minor bands disappeared due to a prolonged duration (40 minutes) of soak for each of the two steps. For reasons that are still unclear, different SOD isozymes were observed to have a different resistance to the soaking effect (data not shown).

In the illumination step, the light intensity of the light box is very important. A high light intensity causes a great output of superoxide radical flux from the riboflavin and TEMED, resulting in a rapid darkening of the gel. Therefore, all bands might disappear under high light intensities. Low light intensities are less effective in color development and require more time;moreover, they are suseptible to interference from an environmental light source. Accordingly, 30 mEm-2s-1 was determined to be suitable for the illumination.

In the densitometry, 100 micron per pixel size and 12 bits of digital resolution in the scanning system were used. The sample lanes were framed as small as possible. The background of the gels were not even especially at the marginal area of the gel, potentially contributing to a variation in the readings. Ideally, blank frames should be located on the upper and lower parts of the lanes to give a better representation of the background. It is not known whether exposing the gel to the laser beam during the scanning causes change to the staining. Nevertheless, few gels needed to be scanned twice.

Theoretically, measurement by interpolation to a standard curve would not obtain the true values of the samples. But it is convenient to show the relationship. The SOD activity of rice seedling crude extract assayed via the ED scheme was higher than that by using the spectrophotometric method, presumably because the superoxide radical flux in the two methods is different. Crude extract of papaya leaves showed a similar situation but with closer values. Crude extract of tobacco leaves, in contrast, showed a different picture. Electrophoretic patterns showed that there were light yellow smears, presumably interference molecules, at the bottom of the tobacco lane and to a lesser extent in the papaya lane, while such a phenomenon was unobserved in the rice lane. Activity assay with the spectrophotometric method showed that the crude extract of tobacco leaves was able to increase the absorbance in the reaction system in enzymatic and nonenzymatic ways without the addition of xanthine oxidase. This suggested that some molecules in the crude extract could initiate the superoxide radical generation system. Extract from papaya leaves showed a similar situation, but to a lesser extent while no such reaction was observed in rice seedling extract (data not shown). The two cases indicated that the crude extract of tobacco leaves contained the largest amount of interfering substances in the three samples, resulting in the largest over-estimation of the SOD activity by the spectrophotometric method, followed by the crude extract of papaya leaves. Rice seedlings contained few interfering substances in the SOD activity assay, resulting in minimal over-estimation. In the spectrophotometric method, these interferences were added to the genuine SOD activity.

The expression of SOD genes are involved in many life aspects including developmental course and in response to environmental stress. Most work on understanding the expression regulation of SOD genes based on RNA gel blot analysis has suffered from the lack of a convenient method to assay SOD activity. With the ED scheme, not only the total SOD activity but also the activity of a single isozyme can be determined efficiently. This technique can provide researchers with an eye to see how a SOD gene is expressed at the enzyme activity level.

Acknowledgments. This work was supported in part by the National Science Council of the Republic of China, under Grant No. NSC 83-0211-B-002-293. The authors are grateful to Dr. Chia-Yin Tsai for helpful discussion and critical review of this manuscript.
              

Chen and Pan — Assay of superoxide dismutase activity

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