When running a classical SOD (superoxide dismutase) assay, samples with xanthine (X) and xanthine oxidase (XOD) produce consistently low absorbance compared to the standards (X+XOD+SOD at different concentrations) and test samples. The time-resolved absorbance curve indicates about 100% OD increase over 30 min, but the values are still the lowest compared to all other samples-standards. Most probably, some of the reagents (X, XOD, SOD or cyt c) went bad. Are there any other explanations? What would be the smartest way to test what substance of these 4 needs replacement?
Please consider another explanation. The tissue/cells/organism you use for SOD analysis can have low levels of enzyme activity.
Yes, if there were no standards involved, I would consider this. But what we see is the reverse order for the standard dilutions - the most concentrated are on top when curves of absorbace run over time are plotted (they consequently have higher slopes for the regressions vs. SOD concentration). Uninhibited control is the lowest...Everything is inperfect order, just reverse:)
Sometimes the reaction among xanthine, xanthine oxidase (tha reacting produce O2-) and cyt is fast. You can try with a shorter time for reading OD.
I have always performed the SOD activity assay considering the increase of absorbance within 2-3 minutes, not more. The reaction is quite fast indeed. Maybe you could also try to dilute a bit more the XOD.
Thank you Estibaliz, this is exactly what we observe too. 4 min is max... Which makes it a bit difficult to get a good slope, the shorter period you select, the higher will be OD-time slope. Do you have any suggestion how to get around this?
Sorry for not answering earlier, Elena! I have not entered this website for a while... Well, what we do to calculate enzymatic activity is use the value of the increase of absorbance from t=0 to t=2 min (At2-At0) and transform this value into a percentage considering that 100% SOD activity is the increase of absorbance reached by our control. We perform nitration assays so SOD activity decreases when it is nitrated and our control is the non-nitrated enzyme. I am not sure of this is what you were asking... Just let me know.
Thank you, but I was not perhaps clear in my question. The shorter incubation time – the higher slope is observed. According to the SOD protocol, the time period for calculations should be selected using the linear range with the highest slope time for the uninhibited reaction. In this case, the highest slope will be observed during the period that is so short that it is not feasible to run the assay for (i.e., the time to measure all wells will be longer than the optimal reaction time). How to get around this problem? ANother issue here is that number of point measurements to calculate slope will be too low for any regression (2-3?).
Are you using just increase in absolute absorbance values from t=0 to t=2 and no slope-based calculations? Could you point me to a refence describing this calculation method?
What do you mean by "incubation time"? What do you incubate? You must try to anotate de interval of absorbance of 0.100/0.120. As I said, this is often obtained within 60-90 seconds. This interval usually gives a pretty lineal measurement. You should make a decision on what range you think is considerably lineal and perform all measurements the same way. The concentration of XOD has to be adjusted in order to obtain that range of absorbance range in that timing. I base my calculations on FMcCord and Fridovich (J. Biol. Chem., 1969, see Fig. 6). You can also check Larrainzar et al. (Meth. Enzymol., 2008).
Thank you. By "incubation time" I mean the 60-90 sec that you are referring to. "Pretty linear" is what is bothering me, because the linearity improves with shortening of this time and it is difficult to "make a decision on what range is considerably linear" because the number of time points with actual absorbance measurements gets fewer. I was also confused by your answer that "use the value of the increase of absorbance from t=0 to t=2 min (At2-At0) and transform this value into a percentage considering that 100% SOD activity is the increase of absorbance reached by our control" but now I see what you mean and as long as the t is the same for the inhibited and uninhibited samples, this is ok. Do you use Blank (X without XOD) in addition to the uninhibited (X+XOD)? If you do, how much variation do you see there?
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