The Effects of pH, Temperature, and Enzyme Concentration on the Catalytic Rate
of Alkaline Phosphatase (ALP)
Bio 1
3/28/2022
I. Abstract
The purpose of this experiment was to determine the effects of not only pH and
temperature, but enzyme concentration on catalytic rate alkaline phosphatase (ALP). Three
experiments were conducted; the first placed a high enzyme concentration of ALP into three
solutions of varying pH balances with the substrate para nitrophenol-phosphate (pNPP) and had
one controlled solution containing just the substrate that were put into a spectrophotometer.
Absorbance in the solutions after exposure to these environments for five minutes was used to
evaluate the enzyme activity of ALP in different pH conditions compared to the controlled
environment and observe what happens to enzymes as they are placed in acidic, neutral, and
basic pH environments. During this experiment, a hydrolysis reaction was invoked, where pNPP,
a clear solution, turned into pNp, a yellow solution. Through the hydrolysis reaction, it was
shown that the optimal pH for ALP was neutral (2.77E-04 A/s). The second experiment was used
to determine the effect of temperature on the catalytic rate of ALP. The cuvettes were filled with
a mixture of the substrate, pNPP, and an Alkaline buffer as the microcentrifuge tube was filled
with a low enzyme concentration of ALP and placed into four environments of different
temperatures for twenty minutes. It was found that the optimum temperature for ALP was 23°C
, (U= 2.92E-04). Lastly, the third experiment was used to determine the effect of enzyme
concentration on the catalytic rate of ALP. The cuvettes were filled with the substrate mixture
and each cuvette with a different level, ranging from low to high, of ALP concentration and then
put into the spectrophotometer every 30 seconds for five minutes. The absorbance recorded
showed as the enzyme concentration increased, so does the rate of reaction. These experiments
showed that pH, temperature, and enzyme concentration do have an effect on enzyme activity.
II. Introduction
From the moment we are created, chemical reactions are happening all around you and inside
your body. Enzymes are an essential part of life and crucial in order to stay alive; they help
catalyze all kinds of chemical reactions essential to life, such as reproduction. An enzyme is
defined as a protein-based catalyst that increases the rate of reactions (Ferrall-Fairbanks et al.,
1970). Enzymes engage in chemical reactions by forming temporary bonds with one or more
reactant molecules, and molecules of the substance called substrates bond to the active site on the
enzyme (Castro, 2014). The binding of the substrate to the enzyme causes the catalysis of the
reaction, resulting in the rate of the reaction speeding up. An enzyme is not required for a
reaction to occur, but many reactions that would take years in the absence of catalysis can occur
in fractions of seconds (Cooper, 1970). The enzyme alkaline phosphate (ALP) can be
categorized as a “marker enzyme” in the sense of its functions being used to diagnose a variety
of diseases. ALP is an enzyme that catalyzes the hydrolysis of a phosphate group attached to
proteins or lipids in vivo (Nyugen et al., 2021). With the presence of the in vitro substrate para
nitrophenol-phosphate (pNPP), ALP and pNPP interact through the hydrolysis of the phosphate
which yields pnPP (colorless) and turns yellow (Nyugen et al., 2021). This experiment was used
of Alkaline Phosphatase (ALP)
Bio 1
3/28/2022
I. Abstract
The purpose of this experiment was to determine the effects of not only pH and
temperature, but enzyme concentration on catalytic rate alkaline phosphatase (ALP). Three
experiments were conducted; the first placed a high enzyme concentration of ALP into three
solutions of varying pH balances with the substrate para nitrophenol-phosphate (pNPP) and had
one controlled solution containing just the substrate that were put into a spectrophotometer.
Absorbance in the solutions after exposure to these environments for five minutes was used to
evaluate the enzyme activity of ALP in different pH conditions compared to the controlled
environment and observe what happens to enzymes as they are placed in acidic, neutral, and
basic pH environments. During this experiment, a hydrolysis reaction was invoked, where pNPP,
a clear solution, turned into pNp, a yellow solution. Through the hydrolysis reaction, it was
shown that the optimal pH for ALP was neutral (2.77E-04 A/s). The second experiment was used
to determine the effect of temperature on the catalytic rate of ALP. The cuvettes were filled with
a mixture of the substrate, pNPP, and an Alkaline buffer as the microcentrifuge tube was filled
with a low enzyme concentration of ALP and placed into four environments of different
temperatures for twenty minutes. It was found that the optimum temperature for ALP was 23°C
, (U= 2.92E-04). Lastly, the third experiment was used to determine the effect of enzyme
concentration on the catalytic rate of ALP. The cuvettes were filled with the substrate mixture
and each cuvette with a different level, ranging from low to high, of ALP concentration and then
put into the spectrophotometer every 30 seconds for five minutes. The absorbance recorded
showed as the enzyme concentration increased, so does the rate of reaction. These experiments
showed that pH, temperature, and enzyme concentration do have an effect on enzyme activity.
II. Introduction
From the moment we are created, chemical reactions are happening all around you and inside
your body. Enzymes are an essential part of life and crucial in order to stay alive; they help
catalyze all kinds of chemical reactions essential to life, such as reproduction. An enzyme is
defined as a protein-based catalyst that increases the rate of reactions (Ferrall-Fairbanks et al.,
1970). Enzymes engage in chemical reactions by forming temporary bonds with one or more
reactant molecules, and molecules of the substance called substrates bond to the active site on the
enzyme (Castro, 2014). The binding of the substrate to the enzyme causes the catalysis of the
reaction, resulting in the rate of the reaction speeding up. An enzyme is not required for a
reaction to occur, but many reactions that would take years in the absence of catalysis can occur
in fractions of seconds (Cooper, 1970). The enzyme alkaline phosphate (ALP) can be
categorized as a “marker enzyme” in the sense of its functions being used to diagnose a variety
of diseases. ALP is an enzyme that catalyzes the hydrolysis of a phosphate group attached to
proteins or lipids in vivo (Nyugen et al., 2021). With the presence of the in vitro substrate para
nitrophenol-phosphate (pNPP), ALP and pNPP interact through the hydrolysis of the phosphate
which yields pnPP (colorless) and turns yellow (Nyugen et al., 2021). This experiment was used
