Effect of pH on Invertase Activity and Sucrose Assay using DNS Method

Jennelie B. Manaog, Charisse V. Misola, Chrisha R. Montemayor, Hajime Q. Nakaegawa, Maria Elijah A. Natanawan
Group 5, 2A Biochemistry, Faculty of Pharmacy, University of Santo Thomas

ABSTRACT
Enzymes are biomolecules that speed up or catalyze chemical reactions by lowering the energy of activation. This
experiment involves the use of invertase, an enzyme that can be extracted from bakers yeast. The main objective of
the experiment was to determine the effects of pH in the activity of the enzyme. It involved three parts: (1) the
extraction of invertase, (2) assay of sucrose, and (3) knowing the effects of pH on invertase activity. A chemical
reaction involved was the hydrolysis of sucrose into simpler sugars, glucose and fructose, to react with the DNS
reagent. A red-brown discoloration was observed in the reduction of DNS. A hydrolyzed-sucrose standard curve was
then plotted using the Dinitrosalicylic Method in which the slope of the equation was obtained. Using six different pH
of buffer solutions, the effect of pH was observed on invertase activity. Results show that the optimum pH of the
enzyme invertase was at pH 5.00, where a bell-shaped graph was observed. This means that at this pH, the enzymes
work is at its best.

INTRODUCTION hydrolyze glycosidic (or acetal) linkages with a

substrate specificity for sucrose. The yeast form
Enzymes play an important role in the life of the enzyme has been assigned the unique four
of living organisms. They are biomolecules that digit enzyme classification code (EC) number of
speed up or catalyze chemical reactions. They 3.2.1.26 and it is also commonly called -
maintain and regulate almost all processes in the fructofuranosidase or sucrase. [4]
body. They can actually be utilized without being
used up in the chemical reaction. Like all To purify a specific protein from a mixture
catalysts, enzymes work by providing an that contains many other proteins, it is essential
alternative pathway that requires lower activation to be able to selectively identify and measure the
energy for a reaction than would otherwise be amount of the target protein without interference
required. [1] from others in the sample. [4]

Almost all enzymes are proteins which are Because of this, the use of Sucrose Assay
folded into complex shapes that allow smaller must be done using Dinitrosalicylic colorimetric
molecules to fit into them. The place where these method must be done for the purpose of
substrate molecules fit is called the active site. If comparison. This method tests for the presence
the shape of the enzyme changes, its active site of free carbonyl group (C=O), the so-called
may no longer work because the enzyme has reducing sugars. It involves the oxidation of the
been denatured. [2] aldehyde functional group present in, for
example, glucose and the ketone functional
Different factors can affect enzymatic group in fructose. Meaning, 3,5-dinitrosalicylic
reaction. These will include enzyme and substrate acid (DNS) is reduced to 3-amino-5-nitrosalicylic
concentration, temperature, and pH. Each factor acid under alkaline conditions. [5]
highly affects the enzymes shape, structure and
activity. Invertase splits the disaccharide sucrose
into the monosaccharides glucose and fructose.
Changes in pH alter an enzymes shape. Invertase is inhibited by high concentrations of its
Different enzymes work best at different pH substrate, sucrose. The invertase enzyme has
values. The optimum pH for an enzyme depends optimum activity at 60 C. Its optimum pH is 4.5
on where it normally works. [2] Below or above although it is active between pH 3.0 and 5.5.
the optimum pH, there is a decrease or increase Inactivation of the enzyme begins at 65 C while
in enzymatic activity. Ionic or electrostatic force the enzyme is totally inactivated after 5 minutes
is being disturbed once there is a change in pH in at 90 C. [3]
the reaction.
The experiment involves two objectives:
Invertase is an enzyme that catalyzes the (1) To extract invertase from Bakers yeast and
hydrolysis of table sugar (i.e. sucrose) into a (2) To determine the effects of changes in pH and
much sweeter, equimolar mixture of glucose and temperature on reaction rates of an enzyme
fructose called invert sugar. [4] Its systematic catalyzed reaction. However, this report will focus
name is sucrose glycosidase implying that it is only on the effects of changes in pH on reaction
a member of the subclass of enzymes that rates, and not on the effect of temperature.
EXPERIMENTAL 1 0.25 1.25
A. Compounds Tested (or Samples used) 2 0.50 1.00
3 0.75 0.75
This experiment can be divided into three 4 0.50 1.00
parts, each using different sets of materials and 5 0.25 1.25
reagents. (1) The extraction of invertase from 6 0.00 1.50
yeast involved the use of bakers yeast and
distilled water. After preparation, three drops of conc. HCl
was added and was subjected to boiling water
(2) For the Sucrose Assay, the following bath for 5minutes. Then, 0.15mL of 0.5M KOH
materials and reagents were used: and 2.80mL of 0.1M buffer solution pH 5.00 was
conc. HCl added and mixed thoroughly. Three mL of DNS
Sucrose solution, 10g/L reagent was added after and subjected again into
Distilled water boiling water bath for 10minutes. From here, the
0.5M KOH absorbance was measured using the
0.1M buffer solution pH 5.00 spectrophotometer. A standard curve was later
DNS reagent constructed.

(3) For the effect of pH on Invertase activity, 2. Effect of pH on Invertase Activity

the following materials and reagents were used:
Invertase stock solution Six test tubes were prepared, each containing
DNS reagent 2.90mL of 0.1M buffer solution. Table 2 shows
DNS reagent the pH used for each test tube.
Sucrose solution, 10g/L
0.1M buffer solution pH 1.00 Table 2. pH of 0.1M buffer solutions used
0.1M buffer solution pH 3.00 Test 1 2 3 4 5 6
0.1M buffer solution pH 5.00 Tube
0.1M buffer solution pH 7.00 pH 1.00 3.00 5.00 7.00 8.00 12.0
0.1M buffer solution pH 8.00 used 0
0.1M buffer solution pH 12.00
After preparation, 0.10mL of enzyme
B. Procedure stock solution was added to each test tube and
1. The Extraction of Invertase from Yeast was incubated at 60 C water bath for 5minutes.
Then, 1.50mL of sucrose solution was added and
To extract invertase, 0.25g of bakers yeast again, incubated at 60 C water bath for
was weighed and dissolved into 250mL solution. 5minutes. Three mL of DNS reagent was then
From there, the solution was made to stand for added and immersed in 95 C water bath for
20minutes at room temperature. The supernatant 10minutes to develop the red-brown color. The
was collected through decantation and this will solutions were cooled and the absorbance was
serve as the enzyme stock solution. measured at 540nm. The hydrolyzed-sucrose
standard curve was used to determine the
100mL of enzyme stock solution was amount of sucrose hydrolyzed.
subjected into boiling water bath for 10 minutes.
This solution will serve as the denatured enzyme RESULTS AND DISCUSSION
stock solution.
Unlike other carbohydrates, sucrose is the
2. Sucrose Assay using Dinitrosalicylic only non-reducing common disaccharide. Most
Colorimetric Method tests for sugar detection utilizing such reagents
as Benedict's solution, Fehling's solution, and
Seven test tubes were prepared. Table 1 DNS (3,5-dinitrosalicylic acid) solution result in
shows the corresponding volume in how the mL negative readings for sucrose. However, these
sucrose solution and distilled water were methods can still be applied if sucrose is first
distributed accordingly. hydrolyzed in an acid solution to yield glucose
and fructose. This method is a modification of the
Table 1. Test Tubes used for the Sucrose Assay original DNS method for glucose analysis. [6]
Test Tube mL Sucrose mL distilled
No. standard Solution water
Blank 0.00 1.50
 Figure 1 shows the hydrolysis of sucrose The amount of acid hydrolyzed sucrose in
using water to split the bond between the two each test tube can be calculated by getting the
sugar units. concentration of the enzyme stock solution,
which can be done by dividing 0.25g of yeast by
250mL. Meaning it has a concentration of
1mg/1mL. Using the amount of mL, one can
compute for the amount of mg used then divide it
by 4.50mL, the summation of all volume of
mixtures (namely added 0.15mL 0.5M KOH,
Figure 1. Hydrolysis of Sucrose 2.80mL 0.1M buffer, 0.05mL conc. HCl, and the
1.50mL sucrose solution) placed in the solution.
The Dinitrosalicylic Colorimetric Method is
a method that tests for the presence of a free
carbonyl group, which are the so-called reducing Hydrolyzed-sucrose Standard Curve
sugars. It involves the principle of oxidation of
0.8
the aldehyde functional group present in, for 0.59
0.6 0.52
example, glucose and the ketone functional 0.46
0.41 0.41
group in fructose. Meaning, 3,5-dinitrosalicylic f(x) =0.34
1.13x + 0.21
0.4
acid (DNS) is reduced to 3-amino-5-nitrosalicylic Absorbance (540nm) R = 0.61
acid, as shown in Figure 2. [5] 0.20.05
0
0 0.1 0.2 0.3 0.4
Concentration, mg/mL

Graph 1. Hydrolyzed-sucrose Standard Curve

Figure 2. Reduction of DNS
The graph was not able to show the direct
Spectrophotometry is a method to proportionality of the amount of sucrose present
measure how much a chemical substance to the absorbance due to some inevitable errors.
absorbs light by measuring the intensity of light
as a beam of light passes through sample Using the Sucrose standard curve, it can
solution. The basic principle is that each determine other concentrations by using the
compound absorbs or transmits light over a slope of the equation generated from the given
certain range of wavelength. This measurement information (as shown in Graph 1). This is
can also be used to measure the amount of a important as Table 4 now shows the effect of pH
known chemical substance. [7] in the absorbance of the solution.

Based from the results of the experiment, Table 4. Effect in changes of pH results
all solutions showed a red-brown discoloration, pH Amount of Acid- Absorbance at
only the blank solution remained yellow (the Hydrolyzed Sucrose 540nm
color of the DNS reagent). This is because it does (mg/mL)
not contain a reducing sugar to react with the 1.00 0.223 0.459 A
DNS reagent to form the 3-amino-5-nitrosalicylic 3.00 0.101 0.321 A
acid. 5.00 0.446 0.710 A
7.00 0.115 0.337 A
Table 3. Results gathered from the DNS Assay 8.00 0.182 0.412 A
Test Tube Amount of Acid- Absorbance at 12.00 0.205 0.438 A
No. Hydrolyzed Sucrose 540nm
(mg/mL) To compute for the amount of acid-
Blank 0 0.052 A hydrolyzed sucrose, you can use the equation
1 0.056 0.456 A generated from the linear regression of the
2 0.111 0.407 A results gathered from the DNS assay. For each
3 0.167 0.342 A pH involved, you can use the slope of the
4 0.222 0.407 A equation to solve any concentration where:
5 0.278 0.520 A
6 0.333 0.585 A y= absorbance at 540 nm and
x = concentration, mg/mL REFERENCES
Graph 2 shows the plotted results of the
effects in the changes of pH. [1] Barron, J. (2014). Enzymes Defined and How
to Buy Them. The Baseline of Health Education

Effect on changes in pH [2] Enzymes:

0.60.45 http://www.bbc.co.uk/schools/gcsebitesize/scienc
e/add_aqa_pre_2011/enzymes/enzymes1.shtml
0.4
0.22
0.21
0.18
0.20.12
0.1 [3] Invertase by Kerry Bioscience Bioinvert.
Amount of Acid-Hydrolyzed Sucrose (mg/mL)
0 Retrieved from:
http://www.ncbe.reading.ac.uk/ncbe/materials/e
10
nzymes/invertase.html
020
pH [4] Timerman, A. The Isolation of Invertase from
Bakers Yeast An Introduction to Protein
Purification Strategies. Retrieved from:
Graph 2. Effect on the changes in pH http://cdn.intechopen.com/pdfs-wm/26595.pdf

Based from the graph, it shows that the

optimum pH of the enzyme invertase is at pH [5] Wang, N.S. Experiment 4A: Glucose Assay:
5.00, meaning the activity of the enzyme is at its By Dinitrosalicylic Colorimetric Method. Retrieved
peak around this pH. from:
http://www.eng.umd.edu/~nsw/ench485/lab4a.h
Extremely high or low pH values generally tm
result in complete loss of activity for most
enzymes. pH is also a factor in the stability of [6] Wang, N.S. Experiment 9D: Sucrose Assay:
enzymes. As with activity, for each enzyme there By Dinitrosalicylic Colorimetric Method. Retrieved
is also a region of pH optimal stability. [8] from:
http://www.eng.umd.edu/~nsw/ench485/lab9d.h
In conclusion, in a chemical reaction, the tm
activity of an enzyme is greatly affected by pH. It
can change the shape of the substrate, or even [7] Spectrophotometry. Retrieved from:
vary the charges as it contains different acidic http://chemwiki.ucdavis.edu/Core/Physical_Chem
and basic groups since they are amphoteric istry/Kinetics/Reaction_Rates/Experimental_Dete
molecules. The stability and solubility can also be rmination_of_Kinetcs/Spectrophotometry
affected as there will be no reaction when the
enzyme and substrate cannot identify each other [8] Introduction to Enzymes. Retrieved from:
in the reaction. http://www.worthington-
biochem.com/introbiochem/effectsph.html