1

Alexis Balderas

Biology Lab 1500

Professor Campos

Sarah Kaldas

5 April 2024

Formal Lab Report: The purpose of this experiment was to the effects of pH, enzyme

concentration, and temperature on the catalytic rate of alkaline phosphatase (ALP).

Abstract:

In this experiment, the purpose was to examine the effects of pH, enzyme concentration,

and temperature on the catalytic rate of alkaline phosphatase or ALP (alkaline phosphatase

enzyme). ALP (alkaline phosphatase enzyme) is found to be involved in digestion for humans,

and clinical tests for certain diseases. The experiment was comprised of three parts: determining

the effect of pH on the catalytic rate of ALP (alkaline phosphatase enzyme), determining the

effect of enzyme concentration on the catalytic rate of ALP (alkaline phosphatase enzyme), and

determining the effect of temperature on the catalytic rate of ALP (alkaline phosphatase enzyme).

In each of these parts, the optimal was found regarding the catalytic rate of ALP (alkaline

phosphatase enzyme) in different pH levels, different concentrations of enzymes, and different

temperatures.
 2

The optimal pH level is neutral since the enzymatic activity was higher compared to

acidic and basic pH. The optimal enzyme concentration was high due to enzymatic activity

increasing as the concentration did. The optimal temperature for enzymatic activity would be

32°C or warm. The optimal temperature was found because when water was colder like 4°C or

23°C enzymatic activity was lower compared to 32°C but when temperatures were higher like

60°C enzymatic activity also decreased.

Introduction:

Within chemistry and everyday life, enzymes are extremely important for many reasons.

First would be that every chemical reaction that occurs inside the cell is catalyzed by an enzyme

(Wilson, et al. 2018). Without enzymes, life would cease to exist. Another reason would be ALP

(alkaline phosphatase enzyme), being used to enhance treatment in medicine. ALP (alkaline

phosphatase enzyme) can be used as biomarkers or indicators of numerous disorders like

excessive skeletal mineralization, Paget’s disease, and tumors (Brichacek, A.L., Brown, C.M.,

2019). Observations of abnormal ALP serum levels have been associated with neurological

disorders like Alzheimer’s disease (Brichacek, A.L., Brown, C.M., 2019). ALP (alkaline

phosphatase enzyme) seems like a promising new therapy for patients with sepsis-associated

AKI (Acute Kidney Injury) by providing anti-inflammatory and tissue-protective effects (Peters

et al., 2014). Enzymes are extremely significant in biology, so that’s why it’s important to know

what can affect enzymatic activity, more specifically how pH, enzyme concentration, and

temperature affect enzyme activity.

In this experiment, the enzyme and substrate that were used are alkaline phosphatase

(ALP) and para nitrophenol-phosphate (pNPP). The reason why these two were chosen was due

to the property that ALP (alkaline phosphatase enzyme) turns to yellowish/clear/colorless pNPP
 3

(para nitrophenol-phosphate) when catalyzed. The change from blue ALP (alkaline phosphatase

enzyme) to yellowish/clear/colorless pNPP (para nitrophenol-phosphate) allows reading of

enzymatic activity by using a spectrophotometer. Variations in pH level, enzyme concentration,

and temperature are recorded, and their changes in absorbance reading over time (minutes) are

also recorded. This collection of data allows for enzymatic activity to be calculated.

The alternative hypothesis for part 1 of the experiment was that ALP (alkaline

phosphatase enzyme) has higher enzymatic activity in a neutral solution compared to an acidic or

basic solution. The null hypothesis was that the enzymatic activity would not be higher if it was

in a neutral solution. The second alternative hypothesis for part 2 of the experiment was that the

catalytic rate for ALP (alkaline phosphatase enzyme) increases as the concentration of ALP

(alkaline phosphatase enzyme) increases. The null hypothesis of part 2 of the experiment would

be that enzyme concentration does not affect ALP’s (alkaline phosphatase enzyme) catalytic rate.

The alternative hypothesis for part 3 of the experiment would be that as the temperature

increased so would the catalytic rate. The null hypothesis for part 3 of the experiment would be

that temperature doesn’t affect the catalytic rate of ALP (alkaline phosphatase enzyme).

Materials and Methods

For this experiment, the solutions that were used were labeled solutions A, B, C, D, E,

and F. Part 1 of the experiment measured the enzymatic activity of ALP (alkaline phosphatase

enzyme) in different pH environments by adding bases and acids into the solution. Solution A is

an Alkaline buffer, solution B is a substrate pNPP (0.004 M para nitrophenol-phosphate),

solution C is enzyme ALP (alkaline phosphatase enzyme) in low concentration, solution D is

enzyme ALP (alkaline phosphatase enzyme) in high concentration, Solution E is 6.5 mL of

 4

solution B (pNPP) mixed with 6.5 mL distilled water (dH2O), and solution F is 15 mL of

solution A (alkaline buffer) mixed with 15 mL of solution B (pNPP).

Solution A and F were not used in part 1 of the experiment due to them containing a

buffer and buffers resisting changes in pH. Part 1 of the experiment used 4 cuvettes: 1 was the

control and only contained 3 mL of solution E (pNPP and dH2O) and 2 mL of distilled water

(dH2O), 2 was acidic and contained 3 mL of solution E (pNPP and dH2O) with 1.9 mL of 0.2 M

HCI and 100 µl of solution D (high concentration ALP), 3 was neutral and contained 3 mL of

solution E (pNPP and dH2O) with 1.9 mL of distilled water (dH2O) and 100 µl of solution D

(high concentration ALP), and 4 was basic, it contained 3 mL of solution E (pNPP and dH2O)

with 1.9 mL of 0.1 M Na2CO3 and 100 µl of solution D (high concentration ALP).

Part 1: Estimating the Optimum pH for ALP Activity

Before adding solution D to the cuvettes, it’s important to check the pH of the solutions

to ensure the experiment is going in the correct direction. A strip of broad-range pH paper on a

clean, dry watch glass must be placed to the side. The board-range pH paper is used and visually

verifies that the pH of the solutions is as stated above. Cuvette 1 is used as a control, so it was

used to blank the spectrometer at 405 nm. 100 µl of Solution D was added with a micropipette to

cuvette 2 then covered with parafilm and mixed/inverted. Once the solution was mixed, it was

placed in the spectrometer and the absorbance reading was recorded and used as the initial value

for a time of 0. The reading of cuvette 2 was taken over the course of 5 minutes with 30-second

intervals in between each new reading. Repeat this process for cuvettes 3 and 4 (Wilson et. al,

2018).

Table 1A. Mixing Instruction for pH Experiment

 5

Cuvette Relative Solution E 0.2 M HCI 0.1 M Distilled Solution D Total

pH Na2CO3 water volume

1 (control) 3 mL - - 2 mL 100 µl 5 mL
-

2 Acidic 3 mL 1.9 mL - - 100 µl 5 mL

3 Neutral 3 mL - - 1.9 mL 100 µl 5 mL

4 Basic 3 mL - 1.9 mL - 100 µl 5 mL

Part 2: Determining the Effect of Enzyme Concentration on Catalytic Rate.

8 cuvettes were labeled 1a, 2a, 3a, 4a, 1b, 2b, 3b, and 4b. Cuvettes 1a, 2a, 3a, and

4a. They will be used in part 3 of the experiment except for cuvettes 1b, 2b, 3b, and 4b which

will be used in part 2 of the experiment. Cuvette 1b, 2b, 3b, and 4b will be made up of 3 mL of

solution F (buffer/pNPP). Cuvette 1b, 2b, 3b, and 4b will go in order of lowest to highest relative

to enzyme concentration. Part 2 of the experiment required cuvette 1 from part 1 to be used as a

blank for the spectrophotometer. Before mixing solution C into any of the cuvettes ensure the

spectrophotometer is blanked using cuvette 1 from part 1. Once ready to start measuring catalytic

rate based on different enzyme concentrations quickly mix in 100 µl of solution C (low ALP

concentration) into 1b and place into the spectrophotometer to get initial readings. All cuvette

readings are recorded over 5 minutes with 30-second intervals for new absorbance readings. 2b

will be mixed with 400 µl of solution C (low ALP concentration) and repeat the recording

process like 1b. 3b will be mixed with 200 µl of solution D (high ALP concentration) and repeat

the recording process like 1b. 4b will be mixed with 500 µl of solution D (high ALP

concentration) and repeat the recording process like 1b (Wilson et. al, 2018).
 6

Table 2A. Mixing Table for Effect of Concentration on Catalytic Rate

Cuvette Relative enzyme Solution F Solution C (Low Solution D (High

concentration (buffer/pNPP) enzyme ALP

concentration) concentration)

Blank 0 3 mL - -

1b lowest 3 mL 100 µl -

2b Medium 3 mL 400 µl -

3b Higher medium 3 mL - 200 µl

4b Highest 3 mL - 500 µl

Part 3: Determining the Effect of Temperature

Cuvettes 1a, 2a, 3a, and 4a will be made up of 3 mL of solution F (Buffer/pNPP) and 100

µl of solution C (Low ALP concentration). Do not add solution C into the cuvettes instead place

100 µl of solution C into four different microcentrifuge tubes and incubate each

cuvette/microcentrifuge pair at their different temperatures. Cuvette 1a/microcentrifuge pair are

placed in a temperature of 4°C (refrigerator), Cuvette 2a/microcentrifuge pair are placed in a

temperature of 23°C (room temperature), Cuvette 3a/microcentrifuge pair are placed in a

temperature of 32°C (water bath), and Cuvette 4a/microcentrifuge pair are placed in a

temperature of 60°C (water bath). They are to be incubated in their environment for at least 20

minutes. One at a time, each cuvette was taken from its location and mixed with 100 µl of

solution C, wiped down for any condensation that might’ve formed then placed into the

spectrophotometer. Each cuvette was recorded over a 5-minute course with 30-second intervals

to record new readings (Wilson et. al, 2018).

 7

Table 3A. Mixing Table for Effects of Temperature on Catalytic Rate.

Cuvette Temperature (°C) Solution F Solution C (Low ALP

(buffer/pNPP) concentration)

1b 4°C 3 mL 100 µl

2b 23°C 3 mL 100 µl

3b 32°C 3 mL 100 µl

4b 60°C 3 mL 100 µl

For parts 1, 2, and 3 the enzymatic activities were calculated from the data recorded:

Enzymatic activity = Δ Absorbance / Δ Time. The change (difference) in absorbance was

calculated by subtracting the initial absorbance reading from the last absorbance reading. The

Change in time equaled the number of seconds each part of the experiment was recorded for. It

was 300 seconds for parts 1, 2, and 3 (Wilson et. al, 2018).

Results:

Part 1: Estimating the Optimum pH for ALP activity.

Table 1: Average of enzyme activity depending on ph.

Enzyme Activity (A/s)

pH Average SD

Acidic 8.33E-07 1.97E-05

Neutral 3.02E-04 1.50E-04

 8

Basic 1.44E-04 9.96E-05

Table 1: Average enzyme activity depending on pH has taken the average of all enzyme activity

depending on pH from 4 different groups and their data. It’s a combination of 12 different results

with each group having 3 results based on enzymatic activity that occurred over 5 minutes for the

3 different pH groups. The data was calculated by taking the initial readings of enzymatic

activity and the final reading for each group: acidic, neutral, and basic. Subtracting the initial

reading from the final one and dividing the sum by the total time, 300 seconds, or 5 minutes.

Figure 1: Average Enzyme Activity Depending on pH.

3.50E-04
3.02E-04
3.00E-04
Average Enzyme Activity

2.50E-04

2.00E-04

1.44E-04
1.50E-04

1.00E-04

5.00E-05

8.33E-07
0.00E+00
Acidic Neutral Basic

pH (Acidic to Basic)
 9

Figure 1 demonstrates the relationship between changes in average enzyme activity and pH. The

change in average enzyme activity increased as it reached its optimal pH, neutral but decreased

once passed and became too basic. When the high enzyme concentration was placed in an acidic

or basic pH environment, enzymatic activity was greatly hindered by the relative pH. This causes

the data to form a bell curve representing how both sides of the extremes aren’t useful for

increasing enzyme activity.

Part 2: Determining the Effect of Enzyme Concentration on Catalytic Rate

Table 2: Average Enzyme Activity Depending on Enzyme Concentration

Enzyme Activity (A/s)

Enzyme Concentration Average SD

Low 1.32E-04 8.45E-05

Medium 8.05E-04 7.13E-04

Medium-High 1.24E-03 1.09E-03

High 1.64E-03 0.002528884

Table 2: Average Enzyme Activity depending on Enzyme Concentration was calculated by taking

the initial reading for low enzyme concentration and subtracting that from the final reading for

low enzyme concentration. Once subtracted, the sum was divided by the total time of the reading

which is 300 seconds or 5 minutes. Rinse and repeat that process for medium, medium-high, and

high. Once all the different enzyme concentrations had their enzyme activity calculated, that data

was used to make Figure 2: Average Enzyme Activity Depending on Enzyme Concentration.
 10

Figure 2: Average Enzyme Activity Depending on

Enzyme Concentration.
1.80E-03 1.64E-03
1.60E-03
Average Enzyme Activity

1.40E-03 1.24E-03
1.20E-03
1.00E-03
8.05E-04
8.00E-04
6.00E-04
4.00E-04
1.32E-04
2.00E-04
0.00E+00
Low Medium Medium-High High

Enzyme Concentration (low to high)

Figure 2 demonstrates the relationship between changes in average enzyme activity and enzyme

concentration. The change in average enzyme activity increased as it was placed in higher and

higher enzyme concentrations. The optima of enzyme concertation would be high. The data

follows a linear line meaning if there were even higher enzyme concentrations it would cause the

average enzyme activity to increase as well.

Part 3: Determining the Effect of Temperature on Catalytic Rate

Table 3: Average Enzyme Activity Depending on Temperature

Enzyme Activity (A/s)

Temperature Average SD

4°C 3.86E-04 4.86E-04

20°C 6.04E-04 8.84E-04

32°C 1.17E-03 2.15E-03

60°C 1.04E-03 0.003395815

 11

Table 3: Average Enzyme Activity depending on temperature was calculated by taking the initial

reading for 4°C and subtracting that from the final reading for 4°C. Once subtracted, the sum

was divided by the total time of the reading which is 300 seconds or 5 minutes. Rinse and repeat

that process for 20°C, 32°C, and 60°C. Once all the different enzyme concentrations had their

enzyme activity calculated, that data was used to make Figure 2: Average Enzyme Activity

Depending on Enzyme Concentration.

Figure 3: Average Enzyme Activity Depending on

Temperature
1.40E-03

1.17E-03
1.20E-03
1.04E-03
Average enzyme activity

1.00E-03

8.00E-04
6.04E-04
6.00E-04

3.86E-04
4.00E-04

2.00E-04

0.00E+00
4°C 20°C 32°C 60°C

Temperature(°C)

Figure 3 demonstrates the relationship between changes in average enzyme activity and

temperature (°C). The change in average enzyme activity increased as it reached 32°C but

plateaued going higher in temperatures like 60°C. The optimum temperature for average
 12

enzymatic activity would be 32°C because temperatures that were lower or higher had lower

enzymatic activity.

Discussion:

The purpose of the experiment was to test how pH levels, concentration of enzyme, and

temperature of a solution affect the enzymatic activity of the solution. Part 1 of the experiment

focused on how different relative pH levels affected the average enzymatic activity. Table 1 and

Figure 1, showcase how if a solution with a high concentration of ALP (alkaline phosphatase

enzyme) was placed in an acidic or basic environment it hinders the amount of enzymatic

activity. But if the solution with a high concentration of ALP (alkaline phosphatase enzyme) is

placed in a neutral environment there are higher levels of enzymatic activity. The data creates a

bell curve showing how being on either extreme of the pH scale isn’t good for enzymatic

reactions but rather being right in the middle will allow for the solution with a high concentration

of ALP (alkaline phosphatase enzyme) to have more enzymatic activity. “Various environmental

factors can affect the rate of enzyme-catalyzed reactions through reversible or irreversible

changes in the protein structure. The effects of pH and temperature are generally well understood

(Robinson 2015).”

Part 2 of the experiment focused on how low to high enzyme concentrations affect the

enzymatic activity of ALP in its solutions. Table 2 and Figure 2 show that when there’s a greater

increase in enzyme concentration there’s more enzymatic activity from ALP in its solution. This

can not only be seen visually by watching the change of color of the solutions from blue to

yellowish/colorless but also through readings from the spectrometer. The reading from the

spectrometer measures absorbances to see how much light passes, as the solution turns from blue
 13

to yellowish/colorless it allows more light to pass through, increasing the readings. The data

from Figure 2 appears to be linear meaning if there were higher concentrations of enzymes,

enzymatic activity likely would increase as well. The optimal enzyme concentration was high.

“The relationship between enzyme concentration and the rate of the reaction is usually a simple

one. If we repeat the experiment just described, but add 10% more enzyme, the reaction will be

10% faster, and if we double the enzyme concentration the reaction will proceed twice as fast.

Thus, there is a simple linear relationship between the reaction rate and the amount of enzyme

available to catalyze the reaction (Robinson, 2015).”

Part 3 of the experiment focused on how temperature would affect the enzymatic activity

of ALP. Table 3 and Figure 3 show that the optimal temperature for enzymatic activity was at

32°C. Looking at Figure 3, enzymatic activity at 4°C or 20°C was lower compared to 32°C or

60°C. The enzymatic activity at 60°C was lower compared to 32. Although the curve in Figure 3

isn’t as deep as in Figure 1, it still has a bell curve shape meaning that extreme temperatures on

either end won’t help the enzymatic activity of ALP but rather limit it. That Is why 32°C is

considered the optimum temperature. “Determination of optimum temperature is commonly

applied to characterize enzymes. It is based on the graph which shows the change in an enzyme’s

activity with increasing temperature. (Wojcik and Milek, 2016).”

Almost all the alternative hypotheses were right except the one regarding temperature.

The alternative hypothesis was that as temperature increases so would the enzymatic activity.

This was wrong because once solution 4a was soaked at 60°C the enzymatic activity decreased

compared to solution 3a which was soaked at 32°C. All the null hypotheses were incorrect due to

them being based on the idea that none of these conditions and different variables would affect

the enzymatic activity of ALP. “Enzymes are affected by pH and temperature (Robinson, 2015).”
 14

Research into these specific variables and how they affect the enzymatic activity of any

enzyme is well-researched and supported throughout the scientific literature.

 15

References

Wilson, C.; Nguyen, J.; D’Alessio, N.; Lavin, E. S.; & Keith, E. (2018). Properties of enzymes

[Class handout]. Department of Biology, Nova Southeastern University, Davie, FL.

Brichacek, A.L., Brown, C.M. Alkaline phosphatase: a potential biomarker for stroke and

implications for treatment. Metab Brain Dis 34, 3–19 (2019).

https://doi.org/10.1007/s11011-018-0322-3

Peters, Esther, et al. “Alkaline phosphatase: A possible treatment for sepsis-associated acute

kidney injury in critically ill patients.” American Journal of Kidney Diseases, vol. 63, no.

6, June 2014, pp. 1038–1048, https://doi.org/10.1053/j.ajkd.2013.11.027.

Robinson P. K. (2015). Enzymes: principles and biotechnological applications. Essays in

biochemistry, 59, 1–41. https://doi.org/10.1042/bse0590001

Wojcik, M., & Miłek, J. (2016). A new method to determine optimum temperature and

activation energies for enzymatic reactions. Bioprocess and biosystems

engineering, 39(8), 1319–1323. https://doi.org/10.1007/s00449-016-1596-7