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O2k-Network discussion forum: Problems with impurities of catalase preparation and high ammonium in superoxide dismutase (2017-07-04)

Janne Purhonen

I have used catalase in the Amplex Red-HRP assay to assess catalase-sensitive H2O2 production. I found out that my batch of catalase from sigma (C9322) decreases respiration in isolated mouse kidney and heart mitochondria but with liver mitochondria it stimulates respiration (Fig1 and Fig2). With liver mitochondria the increase in respiration was oligomycin and antimycin sensitive. Interestingly, the effect is also genotype dependent. An inquiry to Sigma revealed that this catalase product is a crude preparation with a considerable amount of unidentified impurities. Next, I tried to dialyse the enzyme but it did not fix the problem but I learned that the catalase product contained a water and buffer insoluble fraction, removal of which did not fix the problem. My colleague also tested the catalase without the Amplex Red-assay reagents with similar results. The same effect was also observed with heat denatured catalase implying that it is not the enzyme itself. Have anyone else experienced similar problems and tested different catalase products? Should higher purity catalase be recommended (Mir06 recipe?)?
Figure 1. Inhibitory effect of catalase or its impurities on CI&CII-linked respiration in isolated mouse heart mitochondria. H2O2 injections (100nM) were used to calibrate simultaneous Amplex Red-HRP assay (data not shown).
Figure 2. Genotype and tissue-dependent effect of catalase or its impurities on CI&CII-linked respiration. (A, top) Isolated kidney mitochondria. (B,bottom) Isolated liver mitochondria. A magenta coloured line represents a respirometry trace from a wild type mouse and red line from a mouse with a mitochondrial defect.
Then one more reagent issue I faced was related to superoxide dismutase (Sigma, S8409). The Superoxide dismutase is in 3.8M ammonium sulphate suspension in this product. A final activity of 5 U/ml superoxide dismutase means that a millimolar ammonium sulphate concentration is introduced into oxygraphy chamber! After realizing this and observing that CI-linked respiration was somewhat inhibited in isolated mouse kidney mitochondria, I have dialysed the superoxide dismutase into phosphate bufferโ€ฆ Some old studies show that ammonia affects TCA cycle function and mitochondrial respiration:
Worcel A, Erecinska M. Mechanism of inhibitory action of ammonia on the respiration of rat liver mitochondria. Biochimica et Biophysica Acta 1962;65(1):27โ€“33.
Katunuma N, Okada M, Nishii Y. Regulation of the urea cycle and TCA cycle by ammonia. Advances in Enzyme Regulation 1966;4:317โ€“35.

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