Effects of Change in pH and Temperature on the Reaction Rates of Enzyme-Catalyzed Reaction

Hernandez Martin, Lantion Elina, Legaspi Jhoe Cynder, Liwag James, Mangabat Allison, Melad Mikee
Group 4 2F Pharmacy Biochemistry Laboratory

ABSTRACT

The objective of this experiment was to determine the effects of changes in pH and temperature on reaction rates of an
enzyme-catalyzed reaction. It was shown that the optimum temperature was and the optimum pH for invertase
activity is at pH 5. Glucose assay was also done and it proved that there is a direct relationship between
Absorbance and the amount of glucose hydrolized.

INTRODUCTION

Enzymes are important in living organisms. These
enzymes catalyze chemical reactions present in
living organisms without being changed
themselves. Without enzymes, many of these
reactions would not take place at a perceptible
rate. Enzymes catalyze all aspects of cell
metabolism. This includes the digestion of food, in
which large nutrient molecules (such as proteins,
carbohydrates, and fats) are broken down into
smaller molecules; the conservation and
transformation of chemical energy; and the
construction of cellular macromolecules from
smaller precursors. Many inherited human
diseases, such as albinism, result from a deficiency
of a particular enzyme.

Sucrose, or table sugar is an Organic compound,
colourless, sweet-tasting crystals that dissolve in
water. Sucrose is a disaccharide; hydrolysis, by
the enzyme invertase, yields invert sugar (so
called because the hydrolysis results in an
inversion of the rotation of plane polarized light), a
50:50 mixture of fructose and glucose, its two
constituent monosaccharides. Sucrose occurs
naturally in sugarcane, sugar beets, sugar-maple
sap, dates, and honey. It is produced commercially
in large amounts (especially from sugarcane and
sugar beets) and is used almost entirely as food.


Sucrose + H2O ---> glucose + fructose

Sucrase, also called Invertase, any member of a
group of enzymes present in yeast and in the
intestinal mucosa of animals that catalyze the
hydrolysis of cane sugar, or sucrose, to the simple
sugars glucose and fructose.

Granules of sucrase localize in the brush border (a
chemical barrier through which food is absorbed)
that coats the intestinal villi. If sucrase is absent
from the body there is intolerance to sucrose, and
other sugars (maltose or lactose) must be
substituted for sucrose in the diet to provide
adequate nutrition.

3,5-Dinitrosalicylic acid (DNS or DNSA, IUPAC
name 2-hydroxy-3,5-dinitrobenzoic acid) 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
quantifying carbohydrates levels in blood.

Glucose, also called dextrose, adenosine
triphosphate: members of the four families of
small organic molecules [Credit: Encyclopdia
Britannica, Inc.]one of a group of carbohydrates
known as simple sugars (monosaccharides).
Glucose (from Greek glykys; sweet) has the
molecular formula C6H12O6. It is found in fruits
and honey and is the major free sugar circulating
in the blood of higher animals. It is the source of
energy in cell function, and the regulation of its
metabolism is of great importance (see
fermentation; gluconeogenesis).

EXPERIMENTAL

A. Samples Used
Baker's Yeast
Sucrose Standard Solution, 100mg/L
Conc. HCl
0.5 M KOH
Dinitrosalicylic reagent
0.1M buffer solutions (pH 1, 3, 5, 7, 9, 11)
Glucose Solution, 10g/L

B. Procedures

1. Glucose Assay Using Dinitroslicylic
Colorimetric Method
First, we prepared test tubes as follows:
Groups 1 and 2 each prepared test tubes of 0.25ml
glucose standard solution mixed with 1.25 mL
distilled water;
Groups 3 and 4 each prepared test tubes of 0.5ml
glucose standard solution mixed with 1mL distilled
water;
Groups 5 and 6 each prepared test tubes of 1ml
glucose standard solution mixed with 0.5mL
distilled water;
Groups 71 and 8 each prepared test tubes of 1.5ml
glucose standard solution mixed with 0mL distilled
water.
3 drops(~0.05ml) concentrated HCl was mixed to
each tube and incubated at 90C waterbath for 5
minutes.
Figure1. Waterbath set-up


0.15ml of KOH was then added to neutralize the
solution. 2.80ml of 0.1M buffer solution at pH 5
was then mixed. 3ml of DNS reagent was also
added anfd the test tubes were immersed in 95C
water bath for 10 minutes to develop the
characteristic red-brown color.
The test tubes were then cooled and the
absorbance was measured at 540nm.
The hydrolyzed glucose standard curve was then
constructed by pltting A540 against concentration
in (mg/ml).

2. Effect of pH on Invertase Activity

6 numbered test tubes were prepared. 2.9 ml of
appropriate 0.1M buffer solution. 0.1 ml enzyme
stock solution to each test tube was added and
mixed . All test tubes were incubated in 60 C
waterbath for 5 minutes. 1.5 ml of glucose solution
was then added to each and the reaction mixture
was incubated in 60C waterbath for 5 minutes. 3ml
of DNS reagent was then added and the test
tubes were once again immersed in a waterbath of
95C for 10 minutesto develop the characteristic
red-brown color. The solutions were then allowed
to cool.
Blank solutions were also prepared but this time,
denatured enzyme was added instead of the
enzyme stock solution. Absorbance was then
measured at 540nm. Finally, the amount of
glucose hydrolyzed using hydrolyzed-glucose
stndard curve constructed in the DNS colometric
method.

3. Effect of Temperature on Invertase
Activity

Waterbaths of 20, 30, 50, 60, 70, and 90C were
set up. 6 test tubes each containing 1.5mL glucose
solution were incubated separately for 5 minutes
in each waterbath.
In another test tube, 0.8 ml of enzyme stock
solution was mixed with 19.20 ml of 0.1 M buffer
solution which is of pH 5. An additional of 3ml DNS
reagent was mixed and thtest tubes were immered
in 95C waterbath for 10 minutes to develop the
characteristic red-brown color. Solutions were
then cooled.

RESULTS AND DISCUSSIONS

1. Glucose Assay Using Dinitrosalicylic
Colorimetric Method

This method tests for the presence of free carbonyl
group (C=O), the so-called reducing sugars.
This involves the oxidation of the aldehyde
functional group present in, for example, glucose
and the ketone functional group in fructose.
Simultaneously, 3,5-dinitrosalicylic acid (DNS) is
reduced to 3-amino,5-nitrosalicylic acid under
alkaline conditions:
oxidation
aldehyde group ----------> carboxyl group

reduction
3,5-dinitrosalicylic acid ---------->
3-amino,5-nitrosalicylic acid
Because dissolved oxygen can interfere with
glucose oxidation, sulfite, which itself is not
necessary for the color reaction, is added in the
reagent to absorb the dissolved oxygen.

Table 1. Glucose Assay

Test Tube No. Amount of
Glucose in
mg/mL
Absorbance
540nm

B 0
1 5.56x10
-3
0.207
2 0.011 0.285
3 0.0166
4 0.022 2.54
5 0.0277
6 0.033 2.75



The graph above is the glucose calibration curve. It
is shown here that the amount of glucose is
directly proportional to the absorbance.

2. Effect of pH on Invertase Activity

Enzymes are affected by changes in pH. (Campbell
& Reece, 2002)
Extreme pH values generally result in loss of
activity for most
enzymes. Furthermore, there is a most favorable
pH for enzyme - the
point where the enzyme is most active. This point
is known as the
optimal pH. The aim of this experiment is to find
out the range of pH
which invertase is effective.

Table 2. Effect of pH

Test
Tube
No.
pH Amouunt
of Acid
Hydrolyzed
Sucrose (in
mg/ml)
Absorbance
540nm

1 1 4.3x10
-3
0.044
2 3 4.28x10
-3
0.031
3 5 4.5x10
-3
0.015
4 7 5.18x10
-3
0.129
5 9 5.5x10
-3
0.166
6 11 4.61x10
-3
0.0685

Figure 3. Effect of pH


It is shown in the graph that the peak of
absorbance is seen in pH level 5 meaning that the
optimum pH for invertase activity is at pH 5.

3. Effect of Temperature on Invertase
Activity

Optimum Temperature was obtained at.

Table 2. Effect of Temperature

Test
Tube
No.
T
(C)
Amount of
Acid-hydrolyzed
in mg/ml
Absorbance
540nm
1 20 0.116 0.1875
2 30 0.127 0.0635
3 50 0.137 0.013
4 60 0.180 0.166
5 70 0.140 0.0335
6 90 0.136 0.0115



Based on the results obtained,
REFERENCES
Internet Sources
http://global.britannica.com/EBchecked/topic/47
9680/protein/72580/Role-of-enzymes-in-metabol
ism
http://global.britannica.com/EBchecked/topic/47
9680/protein/72579/Enzymes
http://global.britannica.com/EBchecked/topic/57
1354/sucrase
http://faculty.ksu.edu.sa/aabulhamd/Documents/
II%20lab/GLUCOSE%20ASSAY%20(353).pdf
http://en.wikipedia.org/wiki/3,5-Dinitrosalicylic_a
cid
http://www.123helpme.com/view.asp?id=12070
9
http://global.britannica.com/EBchecked/topic/23
5853/glucose




0
0.5
1
1.5
2
2.5
3
0 2 4 6
Series1
0
0.05
0.1
0.15
0.2
0 5 10
Effect of pH
Absorbance54
0nm
0
0.05
0.1
0.15
0.2
0 0.1 0.2
Effect of Temperature
Absorbance54
0nm