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The Measurement of The Activity of Invertase in Sucrose Solution of PH 8 and PH 12 Using Dinitrosalicylic (DNS) Assay Colorimetric Method PDF
The Measurement of The Activity of Invertase in Sucrose Solution of PH 8 and PH 12 Using Dinitrosalicylic (DNS) Assay Colorimetric Method PDF
ABSTRACT
All living organisms perform biochemical reactions inside their body. To help
accelerate and achieve necessary reaction rates, enzymes catalyse these reactions such
as digestion and metabolism. Enzyme activity is highly selective to specific substrates and
reactions thus, several factors can affect optimal enzyme activity. In this experiment, the
effect of pH on invertase activity was investigated. Samples with mixture of phosphate
buffer, pH 8.0 and 12.0, and invertase in two respective micro test tubes were added
with sucrose. The mixtures were then subjected to water bath prior to the addition of
DNS reagent which produced a red-brown color after a boiling water bath. A blank set-
up was prepared using PB buffer with pH 5.0 and without the addition of invertase.
Reading for absorbance of the samples were done by using a spectrophotometer at 540
nm. Results show that invertase hydrolysed sucrose into reducing sugars, glucose and
fructose, with an absorbance of 0.238, 0.279 and 0.335 in pH 8, 12 and blank samples
respectively. Using the DNS Colorimetric Assay standard curve, the concentration of
reducing sugars was 1.4302x10-3 mol/L for pH 8 and 3.8534x10-3 mol/L for pH 12,
interpreting to enzyme activity which are not optimal nor of standard concentrations in
respective pH levels.
INTRODUCTION Intermediate can also be a reactant
producing an enzyme (Figure 1.)
Enzymes are biological molecules
that affect the speed of different
chemical reactions in all biological
systems and forms of life. They are
essential for having a role in aiding in
digestion and metabolism in the body.
Also, enzymes aid in breaking large
molecules into smaller molecules so that Figure 1. Substrate and reaction
it can be absorbed by the body. Since specificity of Enzymes.
enzymes are highly selective due to the
shapes of the molecules, they catalyze There are several roles of
specific reactions only. Enzymes bind enzymes inside the body and each part
with the molecules called substrates. of the body has a specific enzyme in it.
Substrates bind to, active site, the region One of these is the Invertase, also called
on the enzyme molecule (Castro, 2014). beta-fructofuranosidase and is produced
The product formed with an enzyme is and purified from baker’s yeast. Acting as
called the reaction intermediate. an immune booster, anti-oxidant,
antiseptic are the primary roles of
invertase in humans. (Kulshresthaa et in oxidation which produce a reddish
al., 2013). This enzyme aid in the brown colored complex, 3,5-
hydrolysis from sucrose to fructose and Dinitrosalicylic acid is reduced to 3-
glucose. Sucrose is composed of an amino-5-nitrosalicylic acid (Figure 3) with
alpha-D-glucose molecule and a beta-D- an absorbance maximum of 540 nm. The
fructose molecule which bonds to alpha- objectives of this experiment are: to
1,4-glycosidic bond and with the determine the effects of changes in pH
presence of the enzyme, invertase, in the on the reaction rates of enzyme-
bond is cleaved due to hydrolysis catalyzed reactions and to measure the
resulting to mixture of glucose and enzyme activity using the Dintrosalicylic
fructose, (Figure 2) wherein the mixture acid (DNS) Assay method.
is called invert sugar (Wang, n.d.).
Absorbance
2 1.4623
1.5 1.1227
1 0.4387
0.1677
0.5 y = 16.92x + 0.2138
0.0953 R² = 0.9176
0
0 0.05 0.1 0.15
Figure 7. Boiling Water Bath for DNS
Concentration
Assay
200 µL of samples prepared were Figure 9. Standard Curve for DNS
then micropipetted into the wells of the Colorimetric Assay.
microplate. The absorbance was then
read at 540 nm using a Figure 10 shows the microplate of
spectrophotometer. (Figure 8.) the prepared invertase enzyme at pH 8
and 12, as the microplate was subjected
in the spectophotometer the following
absorbance readings were obtained
(Table 2.)
A;msio