University of Santo Tomas Faculty of Pharmacy General Biochemistry Laboratory DETERMINATION OF ENZYME ACTIVITY THROUGH GLUCOSE USING DINITROSALICYCLIC

COLORIMETRIC METHOD WITH THE EFFECT OF pH Jane Catherine SP. Villanueva, Edenn Claudine C. Villaraza, Lorenz Oliver C. Villegas & Cristel Bernice T. Wee Group 10 2G-Medical Technology General Biochemistry Laboratory

ABSTRACT
Enzymes are biological catalysts which can alter or speed up a chemical reaction, without itself being chemically changed at the end of the reaction. In this experiment, invertase which breaks existing bond in sucrose to form glucose and fructose was extracted from Bakers yeast and was subjected to different pH of buffer solution . Dinitrosalicyclic acid (DNS) Assay method was utilized to monitor the enzymatic activity of invertase. It was then observed under a spectrophotometer with 540 nm absorbance. A peak known as optimum pH was shown by plotting the concentration of the hydrolyzed sucrose versus pH. Optimum pH is said to be the most favorable pH value or the point where the enzyme is most active. In the experimented invertase, the result of optimum pH was 5.

I. INTRODUCTION
Enzymes are high molecular weight compounds made up principally of chains of amino acids linked together by peptide bonds. They are also antigenic proteinaceous catalysts, which speed up the rate of a biochemical reaction. They reduce the activation energy that is essential for starting any type of chemical reaction. With a low energy requirement for activation, the reaction takes place faster. The overall performance of an enzyme depends on various factors, such as temperature, pH, cofactors, activators and inhibitors. They are denatured by such agents as elevated temperature and extreme pH values. Their physical state and catalytic function depend distinctly upon a number of physical factors such as pH, temperature, and ionic strength. All enzymes are functionally specific to varying degrees. Enzyme activity is the rate of the catalyzed reaction. When it is plotted against either pH or temperature, the curve usually has a peak or the optimum.1

also known as Saccharomyces cerevisiae. Invertase ,-fructofuranosidase, is a yeast derived enzyme that catalyses the hydrolysis of sucrose in which the bonds of the sugar splits into two namely glucose and fructose. It belongs to a class of enzymes known as glycosidases. Some of these enzymes split the bonds while others twist the bonds at the same time. In the preparation of stock solution, invertase was denatured at 95C to stop activation energy. This would enable the determination of the distinct period. Denaturation is the complete loss of organized structure in a protein. It is said to be irreversible and it also involves the disruption of the secondary and tertiary structure without breaking the primary structure. Glucose was used as the substrate of this experiment in which the enzyme acts to. Glucose (6126) is a mosaccacharide sugar which the product of photosynthesis in plants. It is commonly used in foods, medicines, brewing, and wine making as the source of various other organic chemicals. It is also known as D-glucose, D-glucopyranose, grape sugar, corn sugar, dextrose and cerelose. It is the source of energy in the cell function from the circulating free sugar in blood and a major participant in metabolism. Glucose and fructose make up sucrose.3

Figure. 1 Enzyme Activity - Graphical Presentation of Catalyzed and Uncatalyzed Reaction In the experiment, Invertase was the used enzyme that came from Bakers yeast which is

Figure.2 Chemical structure of glucose

Dinitrosalicylic acid also known as D.N.S.A. or 3,5-dinitrosalicylic acid is a yellow reagent used to determine sugar content especially glucose. It is an aromatic compound that reacts with reducing sugars and other reducing molecules to form 3-amino-5-nitrosalicylic acid, which absorbs light strongly at 540 nm. . It was first introduced as a method to detect reducing substances in urine and has since been widely used, for example, for quantificating carbohydrates levels in blood. It is mainly used in assay of alphaamylase. However, enzymatic methods are usually preferred to DNS due to their specificity. In the DNS assay, the rate of reaction was monitored colorimetrically by measuring the amount of reaction products that reacts with DNS reagent. DNS reagent contains dinitrosalicylic acid, which stabilizes the red color, NaOH that increases the reactivity of sugars and changes the pH of the reaction vessel halting the invertase reaction.3

The extraction of invertase was prepared by dissolving 0.25 grams of Bakers yeast in distilled water to make a 250-mL solution. The mixture was allowed to stand for 20 minutes at room temperature before collecting the supernatant which served as the enzyme stock solution. 2. Preparation of Denatured Invertase Stock Solution Denatured Invertase stock solution was prepared by incubating 100 mL of enzyme stock solution in a boiling water bath for 10 minutes then cooled. The supernatant was then collected to serve as the denatured enzyme stock solution that will be used for the succeeding experiments. 3.Sucrose Assay Using Colorimetric Method Dinitrosalicylic

Fig.3 Chemical structure of 3,5dinitrosalicylic acid The objectives of the experiment were to extract invertase that breaks existing bond in sucrose to form glucose and fructose from Bakers yeast and to determine the effects of the change in pH on the reaction rates of an enzymecatalyzed reaction.2

II. METHODOLOGY
A. Reagents and Materials used Bakers yeast 100 mg/ L Sucrose (Glucose) Standard Solution Conc. HCL 0.5 M KOH DNS reagent 0.1 M Buffer solutions of pH 4,5,6,7,7.8,10 Enzyme Stock Solution Test tubes Hot plate Pipettes Volumetric flask UV-Vis spectrophotometer B. Procedure 1. Extraction of Invertase from Yeast

To construct the hydrolyzed-sucrose curve at against concentration (mg/ml), six test tubes shown in the table bellow were prepared with specific amounts of glucose standard solution and distilled water covered with marble or paraffin film to prevent evaporation of solvent. Afterwards, 3 drops (~0.05 ml) of concentrated HCl was added to each tube. After which they were incubated at 90C water bath for 5 minutes to hydrolyze sucrose. The solution was neutralized by adding 0.15 mL of 0.5M KOH. A 2.80 mL 0.1M buffer solution was added and was mixed using the Vortex mixer. Then, 3 mL of DNS reagent was added. The test tubes were then immersed in a 95C water bath for 10 minutes; doing so will develop the characteristic red-brown color. Allow the mixture to be cooled. After cooling, the absorbance was then measured at 540 nm and the hydrolyzed-sucrose curve was constructed by plotting against concentration. Table1. Tube Preparation For Sucrose Assay Using Dinitrosalicylic Colorimetric Method Tube no. mL of 1% Glucose Standard solution 0 0.15 0.30 0.45 0.50 0.75 1.00 mL of distilled water 3 1.35 1.20 1.05 1.00 0.75 0.50

Blank 1 2 3 4 5 6

4 . Effect of pH on Invertase Activity

For the determination of the effect of pH on Invertase Activity, six test tubes containing a total of 2.90 mL of the appropriate buffer solution shown in the table below were again prepared and labeled. After the preparation, 0.10 mL enzyme stock solution was added to each test tube. It was mixed thoroughly and then were all incubated in a 60C water bath for 5 minutes. 1.50 mL of sucrose solution was added and then incubated the reaction mixture again in 60C water bath for 5 minutes. Incubation allows the solution to undergo enzymatic hydrolysis. The red-brown characteristic was developed after adding 3 mL of DNS reagent and after immersing the test tubes in 95C water bath for 10 minutes. The solutions were then cooled. Blank solutions were prepared by following steps aforementioned, after which enzyme stock solution was added and the absorbance was measured at 540 nm. With the values obtained a hydrolyzed-standard curve was constructed using the dinitrosalicylic colorimetric method. Table2. Test Tube Preparation For Effect of pH on Invertase Activity Tube 1 2 3 4 5 6 No. pH of 4 5 6 7 7.8 10 Buffer Solution

Figure 4.Reduction of Sucrose Glucose, and other reducing sugars, reacts with DNS by oxidation reaction producing a colored compound that shows a maximal molar extinction at 540nm. Glucose is reduced into gluconic acid, same as to dinitrosalicylic acid to 3-amino, 5nitro saliccyclic acid, giving the characterisctic red-orange color. Oxidation is a reversible chemical reaction in which one of the reactions is oxidation and the reverse reaction is reduction. Since the absorbance at 540 nm is linearly dependent on the concentration or mass of glucose the reaction can be used for 3 quantification of reducing sugars. In an enzyme assay, in order to be valid, it must satisfy at least three conditions namely activity must be proportional to the amount of enzyme source added; activity must be constant during the time period of the enzyme; and assay must be carried out at saturating substance concentration.4 During an enzyme-catalyzed reaction, in the experiment, invertase was used, the enzyme binds to a substrate to form a complex. Formation of complex leads to formation of transition-states species, which then forms the product. All the invertase, glucose and reagents were allowed to react in the process to be subjected in reading its absorbance or molar extinction Figure 5. Reduction of Glucose

III. RESULTS AND DISCUSSION

A. Sucrose Assay Using Dinitrosalicylic Colorimetric Method

In the experiment, sucrose was supposed to be used as the standard assay unfortunately it did not hydrolyze very well and produce a redorange color when dinitrosalicylic acid was incorporated in the test tube with the presence of heat so instead 1% of glucose was substituted. This then yielded to the desired color and product wanted to be observed. Despite the error in the sucrose assay, correct hydrolyzed sucrose has a principle of breaking its bond and splitting glucose and fructose as seen in figure 4 in the presence of invertase. The result should be a redorange color. This is because the conversion of the 3,5-dinitrosalicylic acid to 3-amino-5nitrosalicylic acid, which contributed to the redbrown color of the mixture.5

Figure 6. Simplest Kinetic Michaelis-Menten Equation

Equation

Absorbance was measured through the use of spectrophotometer which measures the amount of light transmitted through a sample at a given wavelength. The read absorbance of standard test tube was subtracted to blank test tube. This will give only the absorbance of the invertase, without the additional reagents. To compute for the amount of glucose, the equation

 was used. The result observed was shown below.6 Table3. x and y Values for Concentration of 1% glucose and Absorbance at 540nm Concentration of A540 Glucose 0 0 0.25 0.311 0.50 0.618 0.75 0.946 0.83 1.128 1.25 1.350 1.67 2.118

which was 0.012 and m as the slope which was 1.216. The concentration which was being computed for had a variable of x. Using the equation above and substituting the each value to their own specified variable, the amount of hydrolyzed sucrose would then be computed.The table below had shown the computed concentrations perspective to each pH. Table 4. Amount of Acid-Hydrolyzed Sucrose and Absorbance pH Amount of A540 Hydrolyzed Sucrose (mg/mL) 4 0.381 0.476 5 0.455 0.565 6 0.351 0.439 7 0.295 0.311 7.8 0.253 0.320 10 0.090 0.122

2.5

Glucose Standard Curve

y = 1.2264x R = 0.983

2
Absorbane 540 nm

1.5 1 0.5 0 0 0.5 1

Concentration mg/mL

0.6 0.4 0.2 0 0

Effect of pH

1.5

Concentration mg/mL

Figure 7. Glucose Standard Curve After plotting the points using the Microsoft Excel, a linear line or a best fit line could be observed. This mean the process in making a glucose assay as a standard known concentration was done precisely and perfectly. This curve and its values would serve as the standard known concentration which would become the basis on how to get the concentrations used in the effect of pH on Invertase Activity. B. Effect of pH on Invertase Activity To know the effect of the pH on Invertase Activity, the absorbance of each specific buffered pH solution was read with the used of spectrophotometer. This was then employed to get the amount of hydrolyzed sucrose with the interpolation from the standard glucose curve. The formula used to derived each concentration was:

pH

10

15

Fig.8 Amount of Acid-Hydrolyzed Sucrose vs. pH As a result in the effect of pH, the optimum pH observed was correct which was pH 5 because invertase optimum pH ranges form 4.5- 5. The optimum pH also shows the best conformation in which enzyme activity was the highest, meaning the residues of the enzyme was properly protonated and deprotonated. The phenomenon of optimum pH explains that the slight alteration of pH affects the structure thereby altering its function. Also, pH dependence of enzyme activity was a consequence of either acid-base behavior or changing degree of ionization of groups in the enzyme, in the substrate, or in both.5 Figure 8 also shows a bell-shaped curve which indicated a correct graph for the effect of pH found in the invertase.

The variable y was represented by the absorbance of each. While b as the intercept

IV. REFERENCES
From books:
1

Boyer, R. (2006). Concepts in Biochemistry. 3rd ed. New York: John Wiley & Sons, Inc. Campbell & Farell (2009). Biochemistry (7th Ed.). Brooks: Cengage Learning. Crisostomo,A.C.,et.al (2010). Laboratory Manual for General Biochemistry, Quezon City, Philippines: C&E Publishing, Inc. Voet, D., Voet, J.G. (2004). Biochemistry: Biomolecules, Mechanisms of Enzyme Action, and Metabolism. 3rd ed. New York: John Wiley & Sons, Inc

From the Internet:

5

EFFECT OF PH. (2009). Retrieved January,21,2012 from http://www.worthingtonbiochem.com/introBiochem/effectspH.html

6

PH EFFECTS OF ENZYMES (2001). Retrieved January 21, 2012 from http://www.buzzle.com/articles/ph-effect-onenzymes.html