Abstract
Enzymatic catalysis occurs in almost every cells in organism(Alva & Peyton, 2003)s. Although the activity is invisible in many cases, cellular activities involving energy change proves the existence of catalytic function of enzymes. Using tyrosinase which triggers browning effect in potato with its product melanin, one can visualize and compare rate of enzyme activity in different environmental conditions. Same concentration of substrate, temperature, amount of potato tyrosinase were employed, but different pH buffers were offered for each experimental sample. The 5-minute spectrophotometer test for two experimental groups per pH were conducted with substrate-less and enzyme-less controls, and absorbance and change of absorbance for each pH were analyzed. As the pH went up from pH2 to pH10, the rate of enzyme reaction (deltaA/min) got higher. The pH of buffer affects enzymatic activity in that in reasonable range of acidity, the productivity of enzyme gets closer to optimal level, while it is hard to quantify solely the rate of tyrosinase activity in extreme pH conditions because of degradation of substrate catechol which started to turn the sample yellow even before the photospectrometer test.
Introduction
Enzymes are proteins which catalyze biochemical reactions in cells when binding to its substrate by lessening the activation energy of reaction (Biology 1 Laboratory Manual, Swarthmore College, 2017). If the optimal environmental conditions for enzymatic activity are not satisfied, enzymes do not maximize their functions. Especially, when enzymes are stressed with extreme pHs, they become unstable and inactive (Van-Thuoc, Hashim, Hatti-Kaul, and Mamo, 2012).
When quantifying the rate of enzyme activity, one should find way to visualize the change that enzyme causes in the cell. One of the visible enzyme activity is tyrosinase activity which changes skin color of organism by producing melanin pigment when binding to tyrosine, the amino acid which contribute to melanin production in cells (Biology 1 Laboratory Manual, Swarthmore College, 2017). This process can easily be observed from the fruits when they are kept under sunlight through their darkened skin (Iozumi, 1993).
In order to learn how the pH of environment change tyrosinase activity in a vegetables or fruits, the enzyme was extracted from a potato and samples are made with catechol, which mimics tyrosine and bind to tyrosinase, as substrate (Iozumi, 1993). The 5-minute spectrophotometer tests were conducted for each sample with different pH buffer(pH2, 4, 6, 10), with enzyme-less control and substrate-less control for each pH experimental groups. The rate of enzyme activity was measured by spectrophotometer using transmittance of light and then converted into the more detectable result absorbance (Biology 1 Laboratory Manual, Swarthmore College, 2017). One can calculate and compare rate of enzyme activity for each buffer by using the mean slope of absorbance curve of two experimental groups per buffer depending on time. It was expected that as environment divulges from pH6, the rate of tyrosinase activity would decrease because it gets far from the optimal condition of activity.
RESULTS
From 5-minute spectrophotometer test, the absorbance of light of each sample were calculated. The initial absorbance was relatively high in strong acidic and basic conditions (pH2 and pH10), and this led to the overall absorbance of samples with extreme pHs higher than those of pH4 and pH6. The absorbance of light was highest in sample with pH10 buffer (0.291), then pH2 (0.187), pH6 (0.147), and pH4 (0.131) (Fig.1). Substrate-less and enzyme-less controls for each buffer were tested and resulted in nearly zero value of absorbance.
Following the graph of enzyme reaction rate based on different pH (Fig.2), the highest rate of tyrosinase activity occurred in pH10 buffer sample: for pH10, as time goes, the positive slope of rate of change decreased and the graph flattened (Fig.1). On the other hand, for the samples with other pHs, the lines stayed linear relatively. For the rate of tyrosinase activity, as the pH increase from pH2 to pH10, the rate of enzyme activity (DA/min) increased (Fig.2). That is, the absorbance of sample with pH10 buffer changed most quickly, and for pH 2, it changed slowly.
DISCUSSION
Following the result of the experiment, the tyrosinase activity was not optimized when the pH of the environment converges to neutral: the absorbance was higher in extreme pH condition, and the rate of enzymatic reaction got higher following the pH getting higher.
First, for substrate-less control in acid conditions, the initial and final absorbance was measured higher than other controls. That is due to the acidity of buffer. Since enzymes degrade in extreme pH and change their function and structure, the absorbance could be measured high without substrate in acidic buffers (Savada, Hillis, Heller and Hacker, 2017) .
Second, the initial absorbance of tyrosinase sample with pH2 and pH10 were higher than pH4 and pH6 ones. In extreme conditions with a strong acid or a strong base, catechol, the substrate, can be degraded into smaller substances such as succinate and acetyl-CoA so that the browning reaction could have started even before the addition of enzyme in a sample (Alva and Peyton, 2003).
It can also be said that the rate of enzyme reaction of pH10 buffer sample is not really concise. The flattening line for the pH10 experimental graph shows that either substrate or enzyme is damaged and used up. Since the line graph is nonlinear (Fig.1), it is hard to say that the linear regression model is best fit for absorbance of pH10 experimental group over time.Due to these circumstances, the result states that it is difficult to say that the rate of tyrosinase activity is solely quantified through this experiment. Catechol and tyrosinase denaturation blindfolded the actual rate of tyrosinase activity.
Since this study was only focused on tyrosinase in a potato visualized by only spectrophotometer, the future study can be focused on calculating the Gibbs energy, another method of showing enzyme catalysis perceptively, during tyrosinase reaction (Bearne, 2014).