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ABSTRACT
Enzyme activity is affected by several factors. When the enzyme changes in shape and structure, its rate of reaction is
varied. Examples of the things that alters the rate of reaction of the enzymatic activity are pH and addition of
chemicals or reagents. In this experiment, dinitrosalicylic acid colorimetric method and changing in pH were done to
determine the effects of physical and chemical factors to the invertase activity of sucrose. Negative absorbance were
observed after they were tested in the UV-Vis Spectrophotometer.
METHODOLOGY
A. Sucrose Assay By Dinitrosalicylic
Colorimetric Method
A series of test tubes were prepared as
seen in the table below (Table 1). Three
Because dissolved oxygen can interfere with
drops of concentrated HCl was added to
glucose oxidation, sulfite, which itself is not
each tube. The tube was mixed then
necessary for the color reaction, is added in the
incubated in a 90oC water bath for 5
reagent to absorb the dissolved oxygen.
minutes. Then, 0.15 ml 0.5 M KOH was
added to neutralize the solution. A 0.1 M
The above reaction scheme shows that one
buffer solution about 2.8 ml was then
mole of sugar will react with one mole of 3,5-
added and mixed. Three ml of DNS
dinitrosalicylic acid. However, it is suspected that
reagent was put in the mixture. The test
there are many side reactions, and the actual
tubes were afterward immersed in 95 oC
reaction stoichiometry is more complicated than
water bath for 10 minutes to develop the
that previously described. The type of side
characteristic red-brown color. The test
reaction depends on the exact nature of the
tubes were then cooled and their
reducing sugars. Different reducing sugars
absorbance were measured at 540 nm.
generally yield different color intensities; thus, it
is necessary to calibrate for each sugar. In
addition to the oxidation of the carbonyl groups
in the sugar, other side reactions such as the
decomposition of sugar also competes for the
availability of 3,5-dinitrosalicylic acid. As a
consequence, carboxymethyl cellulose can affect
Table 1. Test Tube Preparation For Sucrose Assay Using Dinitrosalicylic Colorimetric Method
y=absorbance
b=intercept
m=slope
REFERENCES
1
PH EFFECT ON ENZYMES. (n.d.).
Retrieved January 18, 2011, from
http://www.buzzle.com/articles/ph-effect-on-
enzymes.html
2
ENZYME ACTIVITY. (n.d.).
Retrieved January 18, 2011, from
http://www.rpi.edu/dept/chem-eng/Biotech-
Environ/IMMOB/enzymeac.htm
3
Tan, Sue Teresa. HOW TO USE DINIT-
ROSALICYLIC ACID
Retrieved January 18, 2011
http://www.ehow.com/how_5221277_use-
dinitrosalicylic-acid.html#ixzz1BIa2r4fx
Table5. x and y Values for Effect of pH on Invertase Activity
pH (x) Amount of Acid-Hydrolyzed 4
Wang, N.S. GLUCOSE ASSAY
Sucrose (y) BY DINITROSALICYLIC COLORIMETRIC METHOD
2 0.218
Retrieved January 18, 2011, from
3 0.033
7 -0.0093 http://www.eng.umd.edu/~nsw/ench485/lab4
7.5 0.0093 a.htm
11 0.09