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It is hypothesized that a greater concentration of product is achieved through an increased substrate concentration. P-international (p-an) is produced from An-Benzene-ODL-Argentine p-international hydrochloride (PAPA) with the aid of the enzyme trying. Analyzing the initial reaction rates that were calculated from p-an concentration in different test tubes which contained varying concentrations of PAPA the relative contribution of substrate availability on initial reaction rate was accessed.
Initial reaction rates significantly increased at higher initial concentrations of PAPA. However, at a PAPA concentration above 0. Mm, there was no significant increase in initial reaction rate. This suggests that a higher initial reaction rate achieved at a raised initial PAPA concentration from lower levels was due to increases in reactions that formed p-international. The effects from elevating initial PAPA concentration to 0. Mm and above proved insignificant, which may indicate enzyme saturation.
Luminescence Jenny Shin; Ivan Richard Low Introduction This study investigates the effect of substrate concentration on the initial rate of reaction when its concentration is much greater than the enzyme concentration. It is hypothesized that the initial rate of a chemical reaction increases as the substrate concentration becomes more abundant. A possible explanation to this phenomenon could be due to the increased probability of collisions occurring between substrate molecules and its associated enzyme, therefore increasing the number of substrate molecules involved with enzyme activity (Moses and Schultz 2008).
Whereas, a low concentration of substrate will reduce enzyme activity as less number of substrate molecules are available to bind to the active site of enzymes (Moses and Schultz 2008). Therefore, it is proposed that an increase in substrate concentration will increase initial reaction ate through greater enzymatic activity. Materials and Methods The reaction from An-Benzene-ODL-Argentine p-international hydrochloride (PAPA) to p-international (p-an) accelerated by the enzyme trying is utilized as a model to test our hypothesis.
Five test tubes were prepared with differing PAPA concentrations and constant trying concentrations, temperature and PH. Trying concentration in the test tubes was much lower than the PAPA concentration. Initial reaction rate was measured by the amount of p-an produced every thirty seconds for the first five minutes of the reaction. All solutions used in this study have been prepared and brought to room temperature before the beginning of every laboratory session. Trying was obtained from bovine pancreas and all chemicals used were brought in from Sigma, a pharmaceutical company.
Quantitative analysis of transmittance to obtain optical density and p-an concentration was collected from a spectrophotometer. A calibration curve was produced from five calibration tubes for each spectrophotometer used to generate the calibration slope, the k value. A system composed of reference trying concentrations and varying PAPA concentrations as applied to access the relative contribution of substrate availability on initial reaction rate. The k value was utilized to convert optical density (obtained from transmittance), into p-an concentration.
Data Analysis The concentration of p-international was determined by the negative log of (transmittance/ 1 00) divided by the k value. To calculate the initial reaction rates for each trial, the difference between p-an concentration at 2 minutes and p-an concentration at 30 seconds was divided by the time difference (1. 5 min). Although 10-12 data sets were collected, only 9 data sets were chosen at random or data analysis. One of the data sets was incomplete due to missing data collection for 0. Mm of PAPA solution so the average initial rate for 0. Mm PAPA was calculated with only 8 trials. Furthermore, another data set was revised by applying a new formula to replace an erroneous calculation for all p-an concentrations. The average of the initial reaction rates for each of the five different initial PAPA concentrations were calculated from the sum of the initial reaction rates from 9 separate trials (and an exception of 8 trials using 0. Mm PAPA) divided by the number of trials. A significance level of 0. 5 was used to calculate confidence intervals of the average initial reaction rates to create error bars. All statistical functions above were performed through excel. Data analysis determined if range included by confidence interval (i. E. Mean – CLC value through mean + CLC value) does not overlap, so we can be 95% confident that the difference between the rate the formation of p-international is due to differing substrate concentrations rather than random chance or sampling error; we can therefore accept our null hypothesis and consider the difference statistically significant.
If range included by confidence interval does overlap, we can be 95% confident that the rate the formation of p-international is due to sampling error and/or random chance and is not statistically significant. Results Data analysis in Figure 1 indicates a steady trend of increased rate of p- international formation with an increasing amount of PAPA concentration over duration of 1. 5 minutes. To illustrate this inclination, the initial reaction rate rose significantly from 0. 003В??0. Mm/min in a test tube containing 0. Mm PAPA to 0. 006В??0. Mm/min in a test tube containing 0. Mm PAPA. Moreover, the initial rate of formation of p-an in the 0. Mm PAPA tubes are significantly lower than in any other tubes. In the 0. Mm PAPA tube, the initial reaction rate is significantly lower than in the 0. Mm, 0. Mm and 0. Mm PAPA tubes. For example, the initial reaction rate of 0. Mm PAPA is 0. 006В??0. Mommy/min as compared to a much higher rate at 0. 0110В??0. Mommy/min for. Mm PAPA.
However, as PAPA concentration increases to 0. Mm, the initial reaction rate in tubes containing 0. Mm, 0. Mm and 0. Mm PAPA are not significantly different from each other. At 0. Mm PAPA, the initial reaction rate is 0. 001В??0. Mommy/ min which is identical to the initial reaction rate of 0. Mm. Discussion Through the investigation on the effects of substrate concentration on initial reaction rate, we hypothesized that the initial rate of a chemical reaction increases as the substrate concentration increases.
If my hypothesis is correct, then through the protocol using trying, the test tube containing the highest PAPA concentration will have the highest reaction rate and the test tube intonating the lowest PAPA concentration will have the lowest reaction rate. Through data analysis, we observed a significant increase in the rate of formation of p-an with a greater amount of PAPA concentration when comparing the initial reaction rates between the tubes with 0. Mm, 0. Mm and 0. Mm PAPA solution as there is no overlap in confidence intervals between the three means (Figure 1).
However, the confidence intervals of the average initial reaction rate of 0. Mm, 0. Mm and 0. Mm PAPA do overlap and therefore are not significantly different from each other (Figure 1). For example, this is displayed with a mean initial rate of p-an formation as 0. 009В??0. Mommy/min for 0. Mm that overlaps the confidence interval of the initial rate of reaction of 0. Mm which is 0. 0110В??0. Mommy/min (Figure 1). The null hypothesis is supported by significant increases in initial reaction rates with an increase of substrate concentration at lower concentrations.
Although, in this experimental design, any concentration above 0. Mm PAPA began to plateau in initial reaction rates, causing an overlap in confidence intervals which showed insignificance in the proposed trend (Figure 1). A plausible explanation to the results of this study is that a higher substrate concentration increases the frequency of collisions between substrate and the active site of enzymes, which in turn increases the probability of substrate binding to an enzyme (Moses and Schultz 2008).
The enzyme-substrate complex that results upon binding activates enzymatic activity in accelerating the conversion of reactants to products (Moses and Schultz 2008). Although trying concentration was set constant at 5. EYE-mm, which was much lower than initial PAPA concentration for all five test tubes, the readily available PAPA molecules were not sufficient to convert all available enzymes to the enzyme-substrate complex (Moses and Schultz 2008). In Figure 1, the initial rate plateau starting at 0. Mm PAPA may be justified by trying saturation (Moses and Schultz 2008).
Therefore, all the trying present were in the midst of a reaction cycle with another PAPA molecule, so free floating PAPA encountering trying were unable to bind to the active site (Moses and Schultz 2008). When this occurs, the initial reaction rate is unable to increase, as the maximal rate is reached (Moses and Schultz 2008). If this experiment continued using a higher concentration of PAPA with an unchanging concentration of trying, the initial rate of formation of p-an will not be significantly different from 0. Mm PAPA due to the saturation of all trying molecules. Trying has a limited number of active sites and a characteristic number of catalytic cycles per unit time, restricting the initial reaction rate to further increase (Moses and Schultz 2008). Conclusion It has been demonstrated that an increase in substrate concentration increases the initial rate of a chemical reaction by utilizing the model reaction of the abstract An-Benzene-ODL-Argentine p-international hydrochloride converted to the product p-international by the enzyme trying.
The results from this study support our null hypothesis, but only limited to lower substrate concentrations. At higher substrate concentrations, initial reaction rates cease to increase which indicates a maximum initial reaction rate. Further studies can be conducted to gain a better understanding in the plateau observed with higher substrate concentrations. Literature Cited Moses, C. D. And Schultz, P. M. (2008). Principles of Animal Physiology 2nd Edition. San Francisco. Pearson Education Inc.