Abstract
The reduction of nitroblue tetrazolium by superoxide radicals generated from photo-reactive riboflavin has been in use for more than four decades to detect superoxide dismutase (SOD) on nondenaturing polyacrylamide gels. SOD research in medicine and biochemistry has warranted the development of multiple assay variants to overcome specific experimental constraints or to combine the SOD assay with other enzyme assays. Fine-tuning reagent concentrations to effectively visualize bands continue to be a major research obstacle in assay development. Herein we describe a straightforward technique to reliably adjust the background color of polyacrylamide gels without compromising assay efficacy. Low micromolar to low millimolar concentrations of yellow riboflavin can be mixed with the blue of reduced nitroblue tetrazolium to controllably produce blue, purple, yellow-brown, or yellow gel backgrounds. The advantage of this technique is that the assay is not modified by the introduction of new reagents. Quantitative reliability of these alternative stains was assessed by plotting determined band intensity values against known enzyme loads. The correlation (R2) values of trial averages were compared against the average correlation of the standard 0.028 mM riboflavin solution using pooled standard deviation and Student’s T-test at 95% confidence. Assay sensitivity was assessed by comparing lowest possible visible enzyme load of the experimental stains with the 0.028 mM riboflavin standard. No difference in the quantitative reliability was found in any riboflavin concentration. The minimum reliable sensitivity of the assay was found to be 10 ng for each concentration of riboflavin. This technique has already been employed to analyze SOD protein expression levels in extracts of Escherichia coli (Bertrand et al., Med Hypotheses 2012; 78:130-133, 2012; Bertrand & Eze, Adv. Enz. Res., 1: 132-141, 2013).
Highlights
Superoxide dismutase (SOD) is the antioxidant enzyme responsible for eliminating superoxide radicals, ubiquitous in most forms of life
SOD is essential for mitigating cellular oxidative damage: Its absence or abnormal expression has been associated with multiple severe pathologies, including neonatal cardiomyopathy [1], hepatocellular carcinoma [2], age-associated muscular atrophy [3], amyotrophic lateral sclerosis [4], down syndrome [5], hypertension [6], acute respiratory distress syndrome (ARDS) [7], and chronic obstructive pulmonary disease (COPD) [8]
It is remarkable that the minimum reliable sensitivity obtained in the present study (10 ng) is somewhat better than 16 ng originally reported by Beauchamp and Fridovich [9]. These results demonstrate that even dramatic changes in riboflavin concentration do not affect assay sensitivity or band quantitation
Summary
Superoxide dismutase (SOD) is the antioxidant enzyme responsible for eliminating superoxide radicals, ubiquitous in most forms of life. Some variations employ a lower concentration of NBT [10] [11] this study] because the original concentration prescribed [9] would be prohibitively expensive for routine analysis and with larger staining solutions. Another variation of the NBT-based assay includes an assay for the simultaneous detection of catalase on the same gel [10]. A significant research obstacle in the development of these assays is the extensive fine-tuning of reagents required to achieve effective band-background contrast and/or to visualize multiple enzymes (e.g., See reference [10]). A straightforward technique for changing the background color for enzyme visualization would be advantageous to assay developers
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