Lactate dehydrogenase (LDH) is an oxidoreductase found in both eukaryotes and prokaryotes. LDH catalyses the formation of lactate and NAD+ from pyruvate and NADH, during the last step of anaerobic glycolysis. The reaction is also catalysed in the other direction by LDH during the Cori cycle. This reaction can be assayed using spectrophotometric techniques; peak absorbance of NADH is at 340nm, but the peak absorbance of NAD+ is 259nm (Powers, et al., 2007). Enzyme assays are performed to measure the rate of an enzyme-catalysed reaction, known as the enzyme activity. Many factors can affect the enzyme activity (including temperature, pH, substrate concentration), so all conditions apart from the one being quantified should be standardised. The
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By calculating the rate of reaction and studying the effects of varied conditions on the reaction, a great deal can be learnt about the enzyme, including how the enzyme could be inhibited, the catalytic mechanism of the enzyme etc. One of the most well-known ways to look at enzyme kinetics is the Michaelis-Menton equation, which relates reaction rate (V) to concentration of substrate ([S]). Its equation can be used to work out the maximum rate of reaction of the system (Vmax) and the substrate concentration at which the reaction rate is half of Vmax (Km, also known as the Michaelis constant). Michaelis-Menton shows that the rate of reaction is dependent of the rate of formation of the enzyme-substrate complex and rate of formation of the product. This means that in the enzyme assay, the rate of reaction is dependent on the formation of NAD+ from NADH (Scopes, R., …show more content…
Specific activity is useful to know for a protein as it is a good indicator of an enzyme 's purity and quality.
The Bradford assay measures total protein in the assay, and was calculated as 4.2mg/mL. The result was calculated from a set of four concentrations, of which there was a standard deviation of 1.3mg/mL and a standard error of 0.66mg/mL. These are relatively large values, perhaps indicating that there was error during the dilution of 1S. As the big increase in undiluted concentration is between 1S2 and 1S4, this indicates error in the dilution of 1S4 from 1S2. Using just the hand-drawn calibration graph to work out the concentrations of the diluted samples did not provide a very accurate result, so I also used the analysis toolpak on excel to work out the equation of the trend lines of the graph, using regression analysis. The concentrations of the diluted samples could then be worked out to a much greater degree of accuracy than by just interpolating them from the hand-drawn
By calculating the rate of reaction and studying the effects of varied conditions on the reaction, a great deal can be learnt about the enzyme, including how the enzyme could be inhibited, the catalytic mechanism of the enzyme etc. One of the most well-known ways to look at enzyme kinetics is the Michaelis-Menton equation, which relates reaction rate (V) to concentration of substrate ([S]). Its equation can be used to work out the maximum rate of reaction of the system (Vmax) and the substrate concentration at which the reaction rate is half of Vmax (Km, also known as the Michaelis constant). Michaelis-Menton shows that the rate of reaction is dependent of the rate of formation of the enzyme-substrate complex and rate of formation of the product. This means that in the enzyme assay, the rate of reaction is dependent on the formation of NAD+ from NADH (Scopes, R., …show more content…
Specific activity is useful to know for a protein as it is a good indicator of an enzyme 's purity and quality.
The Bradford assay measures total protein in the assay, and was calculated as 4.2mg/mL. The result was calculated from a set of four concentrations, of which there was a standard deviation of 1.3mg/mL and a standard error of 0.66mg/mL. These are relatively large values, perhaps indicating that there was error during the dilution of 1S. As the big increase in undiluted concentration is between 1S2 and 1S4, this indicates error in the dilution of 1S4 from 1S2. Using just the hand-drawn calibration graph to work out the concentrations of the diluted samples did not provide a very accurate result, so I also used the analysis toolpak on excel to work out the equation of the trend lines of the graph, using regression analysis. The concentrations of the diluted samples could then be worked out to a much greater degree of accuracy than by just interpolating them from the hand-drawn