This experiment attempted to extract and purify the protein catalase from a bovine liver by following a procedure involving homogenization, ammonium sulfate fractionation, dialysis, a UNO Q-12 FPLC anion exchange column, and a Superdex G75 2.6 X 60 gel filtration column. In total, 45 mL or 895 mg of catalase was obtained over the course of the experiment. The specific activity of the purified catalase was 0.260 units/mg (Table 1).
The final pooled fraction of catalase obtained was 45 mL, a highly significant yield from the original 100.08 g of bovine liver. However, the total amount of catalase obtained was 895 mg. Since it was impractical for approximately 1% of the liver sample used to be catalase, it was concluded that an error was made in early calculations. If the amount of catalase extracted and purified were indeed 895 mg, the results of the SDS-PAGE gel electrophoresis would have produced a large dark blot or band, yet the most pure form of catalase obtained developed one clear band, clearly alike to the pure catalase (Figure 6). An approximation was made that the error was made during the Bradford assay, however not feasible to correct the miscalculation since the final product had been discarded.
Specific activity is the amount of substrate the enzyme converts or reactions catalyzed, defined in terms of enzyme units per mg enzyme protein. An enzyme unit is the amount of substrate converted to product per units of time. The specific activity prior to ultracentrifugation was 2.035 units/mg and decreased to 0.526 units/mg after FPLC anion exchange and 0.260 units/mg after the gel filtration. The reason for the steady decrease in specific activity after every purification step was due to a loss of activity during the 6...
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...5-step procedure involving homogenization, ammonium sulfate fractionation, dialysis and chromatographic procedures including a UNO Q-12 FPLC anion exchange column and a Superdex G75 2.6 X 60 gel filtration column. The protein then was characterized with SDS gel electrophoresis, pH characterization, temperature characterization, and inhibition of enzyme activity. The final sample of catalase was a homotetrameric enzyme, with a molecular mass of 60 kDa, an optimal pH of 6.5, an optimal temperature of 40 °C and was inhibited by copper, cobalt, magnesium, and nickel. By researching catalase and its characteristics in the natural environment of the bovine liver, findings can be related to the mechanisms, structure, and functions of human catalase and studied to find triggers to disease states associated with catalase malfunction and how to alleviate those disease states.
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