Kindly help me proceed to analyse Polyphenol Oxiadse(PPO) enzyme activity in grapes.I have extracted PPO enzyme using Triton-X detergent. (Reference: Kinetic characterisation and thermal inactivation study of polyphenol oxidase
and peroxidase from table grape, the reference is attached).
Polyphenol oxidase (PPO; EC 126.96.36.199) is a copper-containing enzyme, which, in the presence of oxygen, catalyses the hydroxylation of monophenols to o-diphenols (cresolase activity) and the oxidation of o-diphenols to their corresponding o-quinones (catecholase activity)
For analysis of enzyme activity, The protocol for enzyme analysis is as follows:
• The reaction mixture consisted of 1.25 ml of catechol (20mM) in 0.2 M acetate buffer (pH=3.4) and 0.25 ml of freshly prepared enzyme solution.
• The increase in absorbance was measured at 410nm at 30ºC for duration of 5 minutes at every 30 seconds interval.
• The increase in absorbance per time will be used to calculate the enzyme activity.
The enzyme reaction was followed by absorbance measurement of the quinone or derivative, formed as a result of substrate oxidation, at 410 nm for 5 min in a Spectrophotometer.
Time (sec) Abs at 410nm ?A/min
60 0.584 0.027
90 0.608 0.051
120 0.628 0.044
150 0.647 0.039
180 0.664 0.036
210 0.681 0.034
240 0.714 0.05
270 0.728 0.047
300 0.745 0.031
?A/min values were calculated by subtracting the absorbance every 60 secs for 1 minute. I think the average of these values should give the rate of change of absorbance per minute for the complete reaction.
This value obtained by calculation(0.040) is not matching with the slope (0.0007)value obtained in Excel by plotting a curve of linear regression. According to the literature, the values of the slope for a graph of Absorbance plotted against time should be ?A/min.
This value obtained would be velocity or rate of reaction.
This can be converted into enzyme activity by using Beer Lambert's law.
Absorbance = Molar absorptivity (Epselon) * Path length of cuvette * Concentration.
So, Epselon = (Absorbance) / (Path length of cuvette * Concentration).
To find out the constant Epselon, here I want to confirm whether I can take concentration of the substrate (20Millimolar)? Which absorbance should I use for calculating the Epselon?
Further, I have to use this value to calculate concentration of the product formed per time. Hence, by rearranging the equation for Beer Lambert's law,
Concentration = (change in absorbance / min) / (Path length of cuvette *Epselon)
Hence this can be written as,
Concentration = (slope) / (Path length of cuvette *Epselon).
Suppose, this concentration value is obatined as 5 micro mol/min,
the corresponding units for enzyme activity will be 5 units.
I need help in solving all these obstacles in reaching the final result.
In search of an answer,