The Effects of pH, Temperature, and Enzyme Concentration on the Catalytic Rate of the ALP Enzyme 1
The Effects of pH, Temperature, and Enzyme
Concentration on the Catalytic Rate of the ALP
Enzyme
Erdem Arslan
Sarah Koerner
Nova Southeastern University Biology 1500 – DA-5
3/25/2019
I. Abstract
The purpose of this experiment was to determine the optimum pH, temperature and
enzyme concentration levels on the enzyme ALP. Three experiments were conducted. The
first part was finding optimum pH by adding high enzyme concentration (100 µL) to
different pH levels. The optimum pH was found to be neutral (~7) with a corresponding
enzymatic activity of 1.88E-04 U. The second part was finding the optimum enzyme
concentration by adding different enzyme concentrations to the substrate pNPP. The
optimum enzyme concentration was found to be ‘High’ with an enzymatic activity of
0.00375833 U. The third and final part was finding the optimal temperature by adding low
ALP concentration (100 µL) to different temperatures and this experiment yielded to 32°C
being the optimal temperature with an enzymatic activity of 0.00073583 U. The results from
these experiments contribute to knowledge of these optimal conditions which in turn benefits
doctors and scientists as ALP helps diagnose certain diseases in humans.
The Effects of pH, Temperature, and Enzyme Concentration on the Catalytic Rate of the ALP Enzyme 2
II. Introduction
Enzyme activity occurs in almost all living organisms, in fact, human body faces
enzymatic activities daily. These activities are not limited to humans, but also plants,
bacteria, and other living organisms. Enzymes are catalytic proteins that speed up chemical
reactions in the cell of a living organism (Wilson et al, 2019). Thus, it can be interpreted that
enzymes play a crucial role in food digestion. It helps humans digest the food they eat and
absorb energy and nutrients from food. Enzymes do this by increasing the spontaneity of a
reaction, thus lowering the activation energy required to catalyze the molecule (Cuvelier,
2019). In addition, enzymes are extremely important to the medicinal world as they help
diagnose different types of sicknesses and diseases such as bone cancer, jaundice, and
cirrhosis of the liver (Wilson et al, 2019). To better understand the importance of enzymes
and enzymatic activity, this experiment was used to determine how different aspects, such as
temperature, pH, and enzyme concentration, effect the catalytic rate on the given enzyme.
The enzyme used for this experiment was alkaline phosphatase also known as ALP and
the substrate that was used was para nitrophenol-phosphate or pNPP. It is essential to know
both the substrate and the enzyme used before understanding why each of them were used
specifically. ALP catalyzes a phosphate group attached to a lipid or a protein more
effectively when compared to other enzymes. Due to this, it was ideal to use ALP as the
enzyme. Along with the enzyme, an optimal substrate was needed which would yield a color
that would help in observing the change. As a result, pNPP, a colorless substrate, was chosen.
Once the enzymatic activity took place, the pNPP turned yellow because the hydrolysis in the
phosphate yielded to para nitrophenol. The appearance of the yellow substance (para
Commented [EA1]: No personal pronouns, the human
body
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The Effects of pH, Temperature, and Enzyme Concentration on the Catalytic Rate of the ALP Enzyme 3
nitrophenol) was recorded by using a spectrophotometer that displayed the absorbance
(Wilson et al, 2019).
There were three parts to this experiment. The null hypothesis of part one was: Change in
pH will have no effect on the enzymatic activity of ALP while the alternate hypothesis was
Change in pH will have an effect on the enzymatic activity of ALP. For part two the null
hypothesis stated that change in enzyme concentration will have no effect on catalytic rate
while the alternate stated that change in enzyme concentration will have an effect on catalytic
rate. Lastly, the third part’s null hypothesis was: change in temperature will have no effect on
catalytic rate. The alternate hypothesis was change in temperature will have an effect on
catalytic rate
III. Materials and Methods
There were six solutions used for this experiment: Alkaline buffer (Solution A), .003 M
paranitrophenol-phosphate pNPP (Solution B), this was the substrate, low concentration of
Enzyme ALP (Solution C), high concentration of Enzyme ALP (Solution D), solution B
mixed with Distilled water (this was recorded to be solution E). This was used for the pH
experiment (part one). Solution A and B mixed (this was recorded to be Solution F). This
solution was used for both temperature and concentration experiments (parts two and three)
(Wilson et al, 2019).
Part One: Estimating the optimum pH for ALP activity
Solution E was prepared by mixing 6.5mL of distilled water with 6.5mL of solution B
(pNPP). After solution E was prepared, four cuvettes were labeled and prepared as indicated in
Mixing Table 1. A drop of each solution indicated from Mixing Table 1 was placed on a strip of
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The Effects of pH, Temperature, and Enzyme Concentration on the Catalytic Rate of the ALP Enzyme 4
broad-range pH paper that was on a dry, clean watch glass. pH of each solution was recorded. As
shown in Mixing Table 1, Tube 1 was the control tube. Tube 1 was used to blank the
spectrophotometer at 405nm which set the absorbance to 0. Solution D was not added until the
cuvette was ready to be read in the spectrophotometer. After solution D was added, it was
covered with parafilm and inverted to be mixed. This procedure began with cuvette 2. After
solution D was added to cuvette 2 and mixed carefully, the absorbance was recorded every 30
seconds starting from 0 to 5 minutes. The procedure was repeated for cuvettes 2 and 3. The
formula used to find enzymatic activity was dividing change in absorbance over change in time
(Wilson , C. et al, 2019).
Mixing Table 1: Mixing Instructions for pH Experiment
Cuvette
Relative
pH
Solution
E
0.2 M
HCl
Distilled
water
Solution D
High conc.
Total
Volume