DETERMINATION ON THE EFFECT OF TEMPERATURE ON INVERTASE ACTIVITY USING SPECTROPHOTOMETRY Kathleen Adryon Tan, Vienne Czarina Tongco, Chrystel

Marlowe Turla, Carl Jerome Umali, John Henrick Uy, Celina Marie Wong Group 10 2F Medical Technology Biochemistry Laboratory ABSTRACT
In this experiment, the effects of temperature were carefully observed and analyzed to evaluate its effects on invertase activity. Invertase was isolated from Bakers yeast yielding an enzyme stock solution which was used for the procedures. Dinitrosalicylic (DNS) colorimetric method was utilized to measure the activity of invertase. The amount of absorbance at 540nm was measured using a spectrophotometer. After observation and analysis, a sucrose standard curve was constructed by plotting absorbance at 540nm against the concentration (mg/mL). Invertase was then subjected to varying temperatures (20C, 30C, 50C, 60C, 70C and 90C). Spectrophotometry was also performed to get the absorbance at 540nm which was compared to the sucrose standard curve, and the amount of acidhydrolyzed sucrose was calculated. A separate graph was constructed by plotting temperature against amount of acidhydrolyzed sucrose (mg/ml). A peak was observed on the graphs which indicated its optimum temperature. Optimum temperature is the temperature at which the enzyme is most active. Ideally, invertase exhibits high activity over a broad temperature range of 50C-70C with optimum temperature at about 60C. [5]

INTRODUCTION
All living things have the common characteristic of being able to undergo the process of metabolism. Metabolism is defined as the physical and chemical processes that occur in a biological system that are necessary in the maintenance of the vital function required for the conservation of life. The complex process known as catalysis is the key which makes metabolism possible. Enzymes are responsible for the catalysis of biomolecules. Enzymes function as biological catalyst which increases the rate of chemical reaction without being altered in its chemical composition. Enzymes also act selectively by reacting only to one substrate. It performs its function by lowering the activation energy of a particular substrate, thus making it easier to reach product formation. [1] Enzymes are classified according to their reaction mechanism namely: Oxidases (dehydrogenases) for reduction-oxidation reactions, transferases for transfer of functional groups, hydrolases for hydrolysis reactions, lysases for addition of double bonds or vice versa, isomerases for isomerization reactions and ligases (synthetases) for formation of bonds with ATP cleavage. [1] Enzyme activity is affected by two factors namely: pH and temperature which are related to the tertiary structure. Tertiary and quatenary structure are capable of forming pockets (active sites) which are responsible for holding specific substrates. A temperature/pH too high or low can lead to loss of enzyme function (denaturation). [1] Invertase with a systematic name of fructofuranosidase (EC 3.2.1.26) was isolated from Bakers yeast which is classified as a hydrolase. Typically, invertases are enzymes that catalyze the hydrolysis of peptide bonds, specifically the hydrolysis of sucrose into fructose and glucose. Dinitrosalicylic colorimetric method is a test in which dinitrosalicylic acid (3,5dinitrosalicylic acid) is used as a reagent to determine sugar content especially glucose. The DNS technique is employed in order to estimate sugar present in the samples. This is also effectively used in the handling of requirements for clinics in hospital laboratories, taking into account that only a short period of time is required for the process to take place and the reagents are unreactive, low cost and readily available.[4] An instrument known as a spectrophotometer was used to measure the absorbance of the invertase stock solution to be able to be plotted against the values of the amount of acidhydrolyzed sucrose in order to construct a sucrose standard curve which became a basis for the construction of the temperature vs. acidhydrolyzed sucrose curve. [2] Through the proper execution of each method, objectives which included extracting invertase from Bakers yeast and determining the effects of changes in temperature on reaction rates of an enzyme-catalyzed reaction were precisely accomplished.

EXPERIMENTAL A. Compounds tested (or Samples used)

Bakers yeast, Sucrose standard solution (100mg/L), Concentrated HCl, 0.5M KOH, Dinitrosalicylic acid (DNS) reagent, Sucrose solution (10g/mL), Test tubes, Pipettes, Beakers,

Volumetric flasks, Paraffin film, Hot plate, UV-Vis spectrophotometer.

B. Procedure 1. Extraction of Invertase from Yeast

Invertase was extracted by dissolving the 0.25g Bakers yeast in distilled water to make a 250mL solution. The solution was allowed to stand for 20 minutes at room temperature, and then the supernatant was separated from the sediments that settled down that served as the enzyme stock solution.

2. Preparation of Denatured Invertase Stock Solution

100mL of the enzyme stock solution was incubated in a boiling water bath for 10 minutes. The solution was allowed to cool making frothing possible. Only the supernatant was collected that served as the denatured enzyme stock solution.

In a separate test tube, 0.80mL enzyme stock solution with 19.20mL 0.1M buffer solution, pH 5 was added to have a diluted enzyme solution. 3mL of the diluted enzyme solution were added to all the tubes leaving each immersed in its respective water baths for another 5 minutes. A red-brown color characteristic was developed after 3mL of DNS reagent was added to the test tubes and submerged in 95C water bath for 10 minutes. Solution was allowed to cool. Blank solutions were also prepared by the addition of denatured enzyme instead of enzyme stock solution. Absorbance at 540nm were observed and recorded. Amount of acid-hydrolyzed sucrose was computed using sucrose standard curve constructed in the dinitrosalicylic colorimetric method. After the determination of sucrose hydrolyzed, a second graph was constructed with a plot against temperature and acid-hydrolyzed sucrose (mg/mL).

RESULTS AND DISCUSSION

3. Sucrose Assay Using Dinitrosalicylic Colorimetric Method

Series of test tubes were prepared with fixed ration of mL sucrose standard solution to mL distilled water (0:1.50, 0.25:1.25, .50:1.00, 0.75:0.75, 1.00:0.50, 1.25:0.25, 1.50:0). 3 drops of concentrated HCl were added to all test tubes allowing it to be mixed and incubated at 90C water bath for 5 minutes. The solution was neutralized by adding 0.15mL 0.5M KOH. It was followed by the addition of 3mL DNS reagent. The resulting solution was immersed in 95C water bath for 10 minutes to develop the red-brown color characteristic. All the test tubes were covered with marble/cotton during the water bath process to prevent the evaporation of solvent. After the solution has cooled, it was subjected to the spectrophotometer for the determination of its absorbance at 540nm. A hydrolyzedsucrose standard curve was constructed by plotting the absorbance at 540nm against concentration (mg/mL).

Figure 1 Hydrolysis of sucrose Figure 1 shows that sucrose is formed by glucose and fructose connected by a glycosidic bond. Upon subjecting to hydrolysis, components are separated from each other. The reaction occurs at a slower rate, but addition of a catalyst, invertase, made the reaction occur at a rapid rate. Dinitrosalicylic acid was used to detect reducing sugars (free carbonyl group) and other reducing molecules to form 3-amino-5-nitrosalicylic acid under alkaline conditions which strongly absorbed light at 540nm. A red-brown color indicated a positive result. In order for a result to be detectable, a large amount of the sample was required. Test Tube Hydrolyzed sucrose (mg/mL) A540nm Blank 0 0.000 1 0.0033 -0.001 2 0.0067 -0.003 3 0.01 0.019 4 0.013 0.020 5 0.017 0.067 6 0.02 0.069 Table 1 Amount of hydrolyzed sucrose and Absrobance540nm

4. Effect of Temperature on Invertase Activity

Several water baths were laid out with each having varying temperatures (20C, 30C, 50C, 60C, 70C and 90C). 6 test tubes were then prepared containing 1.5mL sucrose solution and incubated separately for 5 minutes in each water bath.

Hydrolyzed-Sucrose Standard Curve 0.08 Absorbance, 540 nm 0.06 0.04 0.02 0 0 0.005 0.01 0.015 0.02 0.025 Concentration mg/mL Figure 2 Amount of hydrolyzed sucrose and Absrobance 540nm curve Amount of hydrolyzed sucrose was computed using the formula C1V1=C2V2 with C1 being the concentration of the sucrose solution which was 0.1mg/mL, V1 being volume of the sucrose standard solution used varying for each test tube, C2 being the unknown and V2 being the total volume of the solution which was 7.5mL. Computations: Amount of hydrolyzed sucrose (mg/mL) of blank test tube: y = 2.8986x R = 0.746 In figure 2, the straight black line drawn was the best representation for the points plotted computed by getting linear regression of the graph using a scientific calculator, but the graph didnt give a linear-like appearance due to some errors committed by the students in performing spectrophotometry. Computations: Slope R2 y 0.403 0.746 2.8986x Amount of hydrolyzed sucrose (mg/mL) of test tube 6:

-0.02

Test Tube

Amount of hydrolyzed sucrose (mg/mL) of test tube 1:

Acid-hydrolyzed sucrose(mg/mL) 1 20 0.0077 2 30 0.0052 3 50 0.0056 4 60 0.021 5 70 0.015 6 90 -0.0048 Table 2 Amount of acid-hydrolyzed sucrose and Absorbance540nm based on temperature Computations:

Temperature(C)

A540nm 0.019 0.013 0.014 0.052 0.036 -0.012

Amount of acid-hydrolyzed sucrose= slope x A540 Amount of hydrolyzed sucrose (mg/mL) of test tube 2: Amount of acid-hydrolyzed sucrose (mg/ml) of test tube 1 (20C):

Amount of hydrolyzed sucrose (mg/mL) of test tube 3:

Amount of acid-hydrolyzed sucrose (mg/ml) of test tube 2 (30C):

Amount of hydrolyzed sucrose (mg/mL) of test tube 4:

Amount of acid-hydrolyzed sucrose (mg/ml) of test tube 3 (50C):

Amount of hydrolyzed sucrose (mg/mL) of test tube 5:

Amount of acid-hydrolyzed sucrose (mg/ml) of test tube 4 (60C):

Amount of acid-hydrolyzed sucrose (mg/ml) of test tube 5 (70C):

Amount of acid-hydrolyzed sucrose (mg/ml) of test tube 6 (90C):

The calculated linear regression equation above was used in order to come up with the value of acid-hydrolyzed sucrose, x, by substituting absorbance540nm to the variable, y, to the equation derived (refer to table 2). Effect of Temperature on Invertase Activity 0.02 Concentration, mg/mL 0.015 0.01 0.005 0 -0.005 -0.01 0 20 40 60 80 100

invertase. Theoretically, the plot should have a bell-shaped graph showing increased enzyme activity with the increase of temperature up until 60C wherein the enzyme activity started to decrease drastically and eventually became almost inactive which is a clear sign of denaturation (refer to figure 4). The curve constructed didnt show the predicted graph based on theory because of some significant errors that included and not limited to: degrees of uncertainty caused by the students due to inexperience usage of the spectrophotometer, instrumental errors caused by the spectrophotometer due to the poor maintenance of the device and unmonitored temperature of the water bath during the experiment which led to higher/lower than what was specified on the instruction given having a big impact on the enzyme activity.

REFERENCES
From books [1] Campbell, M.K. and Farrell, S.O. (2012). Biochemistry. 7th ed. Philippines Cengage Learning Asia Pte Ltd. pg139-145. [2] Crisostomo, A.C., Daya, M.L., de Guia, R. M., Farrow, F. L., Gabona, M.G., Liu, Ma.I.D., Ysrael, M.C. (2010). Laboratory manual in general biochemistry. Quezon City: C & E Publishing, Inc. [3] Skoog, D.A., West, D.M., Holler, F.J., Crouch, S.R., Chen, S.C.(2012). Introduction to Analytical Chemistry. 8th ed. Singapore: Cengage Learning Asia Pte Ltd. From internet sources [4] http://www.ehow.com/how_5221277_usedinitrosalicylic-acid.html (accessed December 30). [5]http://www.invertase.net/single.htm (Accessed December 31). jh

Temperature, C

Figure 3 Temperature and amount of acid-hydrolyzed sucrose curve

Fig. 4 Theoretical effect of temperature on enzyme activity Referring to figure 3, a peak was observed at 60C that indicated the optimum temperature for