Effect of pH on Invertase Activity Alexis Himelo, Hans Hui, Lance Jariel, Hannah Lorraine Lim, Johnvee Magana, Liezel

Mamburam Group 4 2C Pharmacy Pharmaceutical Biochemistry Laboratory ABSTRACT

Determination of the effects of pH on invertase activity is the primary objective of the experiment. Extraction of Invertase from yeast and preparation of Denatured invertase stock solution were performed. Dinitrosalicyclic acid (DNS) Assay method is utilized to monitor the enzymatic activity of invertase., six numbered test tubes were prepared; Invertase was subjected to different pH (4, 6, 7 and 8) of buffer solution and was observed under 540 nm absorbance using spectrophotometer. After observation and analysis, a peak (optimum pH) was observed by plotting absorbance versus pH.

INTRODUCTION
Enzymes are protein formed by the body that acts as a catalyst to cause a certain desried reaction. Enzymes are very specific. Each enzyme is designed to initiate a specific response with a specific result [1]. 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. Invertase, which was used in the experiment, is a yeast derived enzyme which is classified as a hydrolase. Having the official name, -fructofuranosidase (EC 3.2.1.26), it is classified as a hydrolase. Generally, invertase can break peptide bonds and specifically hydrolyzes sucrose to glucose and fructose [2]. Invertase is an enzyme which is usually found in plants. It acts as a catalyst for the hydrolysis of sucrose. Sucrose is a disaccharide composed of glucose and fructose linked by a glycosidic bond, when this bond is cleaved in a hydrolysis reaction, an equal amount of glucose and fructose [3]. 3,5-Dinitrosalicylic is an aromatic compound that reacts with reducing sugars and other reducing molecules to form 3-amino-5nitrosalicylic acid, which absorbs light strongly at 540 nm [4].

Dinitrosalicyclic acid (DNS) Assay is the method used to monitor its enzymatic activity, specifically tackled on the effect of pH on invertase activity. Spectrophotometer was used to determine the absorbance of the solution. The objectives of the experiment are: (1) to extract invertase from Bakers yeast, and (2) to determine the effects of changes in pH on reaction rates of an enzyme-catalyzed reaction.

EXPERIMENTAL
A. Compounds tested (or samples used) For extraction of Invertase from yeast: 0.25 g Bakers yeast, 250 mL distilled water. For preparation of denatured invertase stock solution: 100 mL enzyme stock solution, boiling water bath For Sucrose Assay Using Dinitrosalicylic Colorimetric Method: 6 test tubes with one blank tube: Test tube 1 with 0.25 mL sucrose std. solution, test tube 2 with 0.50 mL, test tube 3 with 0.75 mL, test tube 4 with 1.00 mL, test tube 5 with 1.25 mL and test tube 6 with 1.50 mL sucrose std. solution mixed with certain amounts of distilled water. UV-Vis Spectrophotometer. For Effect of pH on Invertase activity: 6 numbered test tubes, 2.90 mL 0.1M appropriate buffer solution, enzyme stock solution, 60c and 95C water bath, 3 mL DNS reagent, 1.50mL sucrose solution and UV-Vis Spectrophotometer.

Procedure 1.Extraction of Invertase from Yeast Bakers yeast weighing 0.25 g was dissolved in distilled water to make a 250 ml. solution. The solution was allowed to stand for 20 minutes at room temperature. Supernatant was collected as few sedimentation occurred. The supernatant served as the enzyme stock solution that was used for the succeeding experiments. 2.Preparation of Denatured Invertase Stock Solution A 100 ml Enzyme stock solution was incubated in a boiling water bath for 10 minutes. The solution was allowed to cool. Only the supernatant was collected as frothing occurred. This served as the denatured enzyme stock solution which was used for the succeeding experiments. 3.Sucrose Assay Using Colorimetric Method Dinitrosalicylic Enzyme stock solution measuring 0.10 ml was added to each test tube. The solution was mixed thoroughly and was incubated for 5 minutes in a 60 degrees C water bath. A mixture of 1.50 mL of sucrose solution was added. Reaction mixture was then incubated in 60 degrees C water bath for 5 minutes. 3mL DNS reagent was added. The test tubes were immersed in 95 degrees C water bath for 10 minutes to develop the characteristic redbrown color. The solutions were allowed to cool. Blank solutions were prepared by following the steps previously described. Denatured enzyme was added instead, the absorbance was measured at 540 nm. The amount of sucrose hydrolyzed was determined using hydrolyzed-sucrose standard curve constructed in the Dinitrosalicylic colorimetic method.

RESULTS AND DISCUSSION

Dinitrosalicylic acid (DNS) Assay was used to monitor enzyme activity. The principle involved in DNS assay, is that DNS (3,5dinitrosalicylic acid, IUPAC name 2-hydroxy-3,5dinitrobenzoic acid) reacts with reducing sugars (eg. Glucose and fructose) to form 3-amino-5nitosaliclylic acid (ANS). DNS does not react with sucrose because it is a non reducing sugar. Accordingly, the rate of reaction (enzyme activity) is monitored (colorimetrically) by measuring the amount of reaction products (reducing sugars- equimolar mixture of glucose and fructose) that react with DNS reagent. Invertase is classified as a hydrolase, they are able to break peptide bonds and can split sucrose to glucose and fructose Invertase was extracted from Brewers yeast (Figure 1).

To construct the hydrolyzed-sucrose curve at against concentration (mg/ml), six test tubes were prepared with specific amounts of sucrose standard solution and distilled water covered with marble or parafilm 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. 4.Effect of pH on Invertase Activity Six-numbered test tubes were prepared.Afterwards,2.90 ml appropriate 0.1 M buffer solution was added as described below.The test tubes were labeled accordingly. Table 1: The pH of Buffer Solution per Test Tube Tube No. pH of Buffer solution 1 4.0 2 6.0 3 7.0 4 8.0

Figure 1: Reaction of Sucrose and Invertase to Yield Glucose and Fructose The enzyme stock solution and the denatured invertase stock solution (which served as the blank) was used and prepared. The result of test tubes that contains a pH of 4, 6, 7, and 8 was tested in the experiment.

Table 2: pH and Absorbance of Invertase pH 4 6 7 8 Absorbance 0.211 A 0.219 A 0.222 A 0.214 A

[4]Boyer, Rodney (2006) Concepts in Biochemistry 3rd Edition. John Wiley and Sons Inc. U.S.A. [5]Nigam A. and Ayyagari A. (2007). Lab Manual in Biochemistry, Immunology and Biotechnology. McGraw-Hill Publishing Company. India [6]Crisostomo, A.C. et al. (2010). Laboratory Manual in General Biochemistry. C & E Publishing Inc. Philippines. [7]Seager, S.L. and Slaubaugh, M.R.(2011). Organic and Biochemistry for Today 7th Edition. Brooks/Cole, Cengage Learning. U.S.A. [8]http://www.worthingtonbiochem.com/introbiochem/effectsph.html date accessed: January 23, 2012. [9]http://www.buzzle.com/articles/pheffect-on-enzymes.html date accessed: January 24, 2012.

Enzymes are affected by changes in pH. The most favorable pH value - the point where the enzyme is most active - is known as the optimum pH.

Effects of pH on Invertase Activity 0.225 Absorbance 0.22 0.215 0.21 0 5 pH 10

Figure 3: Graph of the Invertase Activity. The rate of a chemical reaction and/or the enzyme activity is greatly affected by the structure of the enzyme. A change in the structure of the enzyme affects the rate of reaction. When pH changes, it leads to alteration in the shape of the enzyme. pH also affect the charge properties and shape of the substrate. Within a narrow pH range, changes in the structural shapes of the enzymes and substrates may be reversible. But for a significant change in pH levels, the enzyme and the substrate may undergo denaturation. In such cases, they cannot identify each other. Consequently, there will be no reaction as such. REFERENCES

[1]Enzymes http://cysticfibrosis.about.com/od/glossary/g/e nzyme.htm [2] http://greenwoodhealth.net/np/invertase.htm [3] http://en.wikipedia.org/wiki/3,5Dinitrosalicylic_acid