Rates of reactions that are catalyzed by enzymes are not constant, and multiple factors could influence reaction rate increasing or decreasing it. Multiple studies report of allosteric regulatory compounds of both protein and nonprotein origin that regulate activity of particular enzyme and thus influence rate of the reaction that is catalyzed by that enzyme. One of the factors that influence rates of enzyme-catalyzed reactions is temperature of the environment in which reaction takes place.
It is supposed that temperature should influence reaction rate in full accordance with the laws of thermodynamics, and increase in temperature should result in increased reaction rate. It is known as well that proteins have tendency to denature when temperature of their local environment increases beyond certain value. Therefore we suggest that increase of enzyme-catalyzed reaction rate will not be infinite, and when increasing temperature will reach denaturation point, the reaction will not proceed anymore or its rate will abruptly drop to the level of uncatalyzed reaction, which for many biochemical reactions is roughly equals to zero.
It is also suggested that concentration of catalyst could influence rate of the reaction, and that increase of enzyme concentration will result in significant increase of reaction rate. The experiment described in the following is intended to check if there is a straight dependence between the concentration of catalytic agent and the rate of reaction catalyzed by that enzyme. The second part of the experiment is intended to check that hypothesis of limited dependence of reaction rate from the temperature of local environment. Materials and methods
In order to perform observations upon influence of enzyme concentration and local temperature on reaction rate, such materials and reactants were used: stock solutions of model enzyme (amylase) of different concentrations (10%, 5%, 2% and 1%), stock solution of starch as enzyme substrate, and iodine solution as indicator of starch presence in the reaction mixture. The following equipment that was used in the experiment: spot plate, test tubes for experimental solutions, a pipet and thermostat which could be set to different temperatures (or water bathes heated to different temperatures).
In our experiment values of 0°C (ice point), 18-25°C (room temperature), 40-50°C, 55-65°C and 100°C (steam point) were used as points of interest. In the experiment that investigated influence of enzyme concentration on reaction rate changes in color of reaction mixtures were monitored over time. Temperature and volumes of substrate stock solution used were identical for all reaction tubes, as well as time of observation. Starch solutions were colored dark blue by adding several drops of iodine solution to enable monitoring of reaction progress.
Equal volumes of enzyme solutions with different concentrations of enzyme were added to the substrate and then color of reaction mixtures was assessed every 15 seconds. In the experiment that was intended to check influence of temperature on the reaction rate, reaction tubes with mixtures of enzyme, substrate (starch) and several drops of iodine solution as indicator of substrate presence was incubated at different temperatures in range from ice point to steam point. Every 20 seconds a drop of 5% amylase solution was added to every reaction tube, and color of reaction mixtures was assessed in the meantime.
Color data obtained at different observation points were compared with data from other reaction tubes obtained in the same observation moment. Results Part 1: enzyme concentration and reaction rate. In the test tube containing reaction mixture with 1% amylase solution, no color change occurred throughout all the time of observation. In the tube with 2% solution, reddish dark brown color was detected at the very end of the experiment. In the tube containing 5% amylase solution, color change was detected at first approximately at the start of a second minute of incubation, and final color of reaction mixture was light brown.
And in the reaction mixture to which 10% amylase solution was added, color changed from dark blue to dark purple at the very beginning of the experiment, and changed to light yellow at the end of incubation, indicating very low or no starch in the reaction mixture. Part 2: temperature and reaction rate. At the freezing temperature no changes were observed over time. Reaction mixture retained dark blue color throughout all observation time. At the room temperature (18-25°C) color started to change at the final minute of observation, and the change was from dark blue to dark reddish purple.
At the temperature of 40-50°C change of color occurred earlier, at the end of second minute of observation, and changes were much more intense from dark blue to yellowish brown. At temperature of 55-65°C dark blue color was changed to reddish brown as early as after 80 seconds of incubation, and at the final observation this reaction mixture had light gold color of diluted iodine solution without any traces of starch. Discussion Amylose is the essential part of starch. In the presence of iodine, solutions of amylose turn dark blue, which is the qualitative test for starch detection.
Digestion of starch by amylase decreases amount of iodine-amylose complexes in the reaction mixture, which in turn decreases intensity of blue color of the solution. The more amylose is digested, the more color of the mixture will change towards the natural yellow color of iodine solution. Comparing color changes in test tubes in the part 1 of the experiment, it is clear that reaction rate was slowest in the first tube, which contained 1% enzyme solution, and the fastest reaction rate was observed in the tube containing 10% solution of amylase.
Mixture inside this tube changed color first among the tubes, and change of color was most intense in the tube with highest enzyme concentration, indicating highest reaction rate and largest degree of amylose digestion in that tube (see Graph 1). Intercomparison of color change rate inside the tubes incubated under different temperatures provided that boiling and freezing environment are unfavorable for amylase, for no changes in reaction mixture colors were observed at all.
However, increase in temperature from the ice point to the room temperature and yet higher resulted in increased color change rate, which indicates higher rate of amylose digestion, i. e. higher rate of enzymatic reaction. Highest reaction rate was observed at the temperature of 55-65°C, which is, consequently, the most favorable temperature for this enzyme, the closest value to its thermal optimum (see Graph 2). Conclusions Amylose catalyzes digestion of starch component amylase. Course of this reaction could be monitored by change of reaction mixture if several drops of iodine solution were added before the reaction started.
Change of color indicates digestion of amylose and iodine release into the solution from amylose-iodine complexes. Reaction rate in excess of substrate depends upon enzyme concentration and is directly proportional to the concentration of the enzyme. Reaction rate is also temperature-dependent: reaction is ceased at the ice point and increases along with temperature increase. Thermal optimum of the amylose is approximately at 55-65°C. When the temperature exceeds that level, thermal denaturation of the enzyme occurs and the reaction rate is slowed down to the level of ice point again.