A well written and structured Biochemistry Practical Portfolio(3704) for 3rd year students constituting of 7 major laboratory experiments namely: Enzyme Kinetics, Bacterial Transformation, Preparation of Plasmid DNA, Lysozyme Assays , Chromatography, Electrophoresis and Applications- Diagnostic an...
, EXPERIMENT 1
KINETIC ACTIVITIES OF
ENZYMES
ABSTRACT
This experiment was done with the aspect of finding the properties and
hydrolyses sucrose to glucose and fructose. Deionised water was used to dilute
(according to the required calculated volumes) the reagents (to avoid
contamination since it’s pure) to provide reactions of different sugar
concentrations which would be used to determine the plotting of the standard
curve and quantification of the product. The data collected and analysed was
used to plot the velocity [Vο] against glucose concentration on Lineweaver-Burk
plot to calculate the Vmax, Km and Kcat.
INTRODUCTION
An enzyme is a biological catalyst which speeds up the rate of reaction without
being altered at the end of the reaction, and its kinetics is the analysis of the
chemical reactions which it catalyses. Analysis or time course of the enzyme
kinetics is mainly based on the quantitative measurement of the rates of enzyme
catalysed reactions and systematic study of the factors that affect these rates.
The rates will determine the characteristics of the enzyme by finding its
parameters: Km (the strength of the enzyme-substrate concentration) Kcat
(turnover number which measures the catalytic production of products under
optimum conditions) Vmax (maximal rate attained when all catalytic sites are
saturates with substrates). For this purpose it is necessary to construct a
mathematical model that embodies the hypothesized mechanisms. Whether or
not the solutions of the resulting equations are consistent with the experimental
data will either prove or disprove the hypothesis. In this experiment, highly
purified methods are practised to enable determination of the parameters,
optimum temperatures are used to avoid denaturation of the proteins and
Page | 3
,preventing misleading results and, finally the conversion of the substrates to
product at a certain rate will determine the enzyme catalytic reactions
Methods & Materials
Reactions were set up, and reagents were added in different glucose
concentration according to the calculations done aseptically in the microtiter
tubes labelled 1-5
Table 1 standard solutions
Volume of Glucose Volume of Volume of
glucose concentration(mM) DNS(µL) Water (µL)
(µL)
1 0 0 10 240
2 1 0.1 10 239
3 2 0.15 10 238
4 3 0.25 10 237
5 5 0.4 10 235
Reaction solutions were made in separate tubes
Table 2 reaction solutions
Reagent (C1) Volume(µL) Final (C2)
Buffer 215
Sucrose 5 0.4
DNS 10
Yeast 20
Final volume 250
After the preparations, the 5 tubes were taken for absorbance
The solutions were loaded onto the microtiter plate using the correct pipette
tips according to the volumes
The plate was taken to the reader and open program to read reaction
kinetics
The enzyme was then added to the plate using an appropriate pipette and
taken for reading
The results were used to calculate V0 at various concentration of sucrose
Km and Vmax were determined using Lineweaver burk plot and the Vmax was
negative.
Many errors were made because students were a bit confused with
calculations and how to operate the program
It took a lot of time to finish this experiment due to inadequate machines for
absorbance reading , so groups had to queue for access
Conclusion
The negative Vmax was probably from:
The human errors during calculations or reading of the absorbance leading
to false results, therefore attention has to be of importance when
conducting experiments
The software settings may have been done wrongly leading to false
readings
The enzyme would have been denatured or contaminated during storage.
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, Figure 2 Lineweaver Burk
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