CP # 4: Initial rate of enzymatically

controlled reactions

• To be able to measure the initial rate of enzyme activity

• To understand why measuring the initial rate is important
• To understand the variables that can affect the rate of an enzyme-catalysed reaction and the
result of changing each variable
• To be able to calculate the rate of a reaction using the gradient of a line
Trypsin
Trypsin
• Milk protein (casein) is broken down by protease enzymes such
as trypsin. The opaque white colour of the milk is replaced by a
clear solution. Light passes more easily through the final solution,
so the reaction can be monitored using a colorimeter .
• There are four variables which can change in this investigation
(IV) : temperature, enzyme concentration, pH or substrate
concentration.
• The procedures for each variable are given in your lab book.
• We will be only doing the Temperature effect.
colorimeter
 Procedure – Part 1: Temperature
• 1.Prepare water baths at the temperatures : 10,20,30,40 and 50 oC. Use thermometers to
ensure each water bath stays at the correct temperature.
• 2. Take 5 test tubes and add 2 cm3 of 1% trypsin solution to each one. Label the tubes with
the temperatures and place each one in the corresponding water bath.
• 3.Take 5 clean test tubes and add 2 cm3 of milk to each one. Label the tubes with the
temperatures and place each one in the corresponding water bath.
• 4.Leave the test tubes for 5 minutes so they reach the required temperature.(to equilibrate))
• 5.Place 2 cm3 of trypsin solution in a cuvette. Use this as a reference cuvette to set the
colorimeter absorbance to zero.
• 6.Pour 2 cm3 of milk suspension at the first temperature being tested into the second cuvette.
• 7.Add 2 cm3 of trypsin solution from the same water bath to the milk in the cuvette. Working
quickly, mix the trypsin solution and the milk by shaking gently, then place the solution into
the colorimeter and start the datalogger.
• 8.Measure the absorbance immediately and then at 15-second interval for 1.5 minutes, or
until there is little change in absorbance.
• 9.Rinse the cuvette with distilled water and repeat steps 6–8 for each of the other four
temperatures. Remember to use the reference cuvette to zero the colorimeter before each
new set of readings. Record the data collected in a results table
Water bath @10oC
•2 cm3 of 1% trypsin solution
•2 cm3 of milk
Water bath @20oC
•2 cm3 of milk
•2 cm3 of 1 % trypsin
Water bath @ 30oC
•2 cm3 of 1% trypsin
•2 cm3 of milk
Water bath @ 40oC
•2 cm3 of 1% trypsin
•2 cm3 of milk
Water bath @ 50oC
•2 cm3 of 1 % trypsin
•2 cm3 of milk
Data logger
 Absorbance/AU

Temperature (0C) 0s 15 s 30 s 45 s 60 s 75 s 90 s
50 1.95 1.56 1.19 0.73 0.56 0.26 0.15

40 1.95 1.42 0.88 0.43 0.24 0.11 0.09

30 1.95 1.17 0.55 0.28 0.14 0.10 0.06

20 1.95 1.56 1.26 0.81 0.49 0.32 0.14

10 1.95 1.80 1.56 1.31 1.06 0.84 0.55

 At temperature 10 C o

Absorbance / AU
Can be calculated
from this graph

This is at 10oC 15 30 45 60 75 90
only
 At temperature 20 C o

Absorbance / AU
Can be calculated
from this graph

This is at 20oC 15 30 45 60 75 90
only
 At temperature 30 C o

Absorbance / AU
Can be calculated
from this graph

This is at 30oC 15 30 45 60 75 90
only
 At temperature 40 C o

Absorbance / AU
Can be calculated
from this graph

This is at 40oC 15 30 45 60 75 90
only
 At temperature 50 C o

Absorbance / AU
Can be calculated
from this graph

This is at 50oC 15 30 45 60 75 90
only
Plot the initial rate against Conclusion
temperature As temperature increases , the
initial rate of reaction first increases.
This is because trypsin and protein
molecules have more kinetic
energy . The random movement of
molecules increases along with the
probability of a successful collision.
Initial rate of reaction / aus-1

More enzyme-substrate complexes

form so the rate of reaction increases.
However, beyond the optimum
temperature, increasing vibrations
break bonds in the enzyme’s
tertiary structure. The active site
changes shape and the enzyme
denatures . The rate of reaction
decreases.
/ oC
 The effect of changing temperature on initial rate of reaction

Dependent variable……………………Initial rate of reaction

Independent variable………………... ..Temperature

Controlled variables………………….. Time to equilibrate

pH of solution (buffer solution),.
Substrate (MILK) concentration and volume
Enzyme(trypsin) concentration and volume

Control…………………………………Experiment without any enzyme

Reliability……………………………...Repeats (5 to 10) for each temperature

Mean, SD , overlap in SD’s

Validity………………………………..control variables, Use of a fit way to measure initial rate of

reaction

Safety…………………………………check previous slides

The effect of changing temperature on rate of reaction
pH
Enzyme concentration
Substrate concentration

Repeat the experiment for each value of IV

Measure IRR for each
Plot on a graph
 Buffer solutions

 The enzymatic reaction itself may change the pH of the solution

e.g. Lipase produces 3 fatty acids and 1 glycerol from triglycerides

 pH influences the rate of reaction of the enzyme

Need to prevent changes in pH in the solution

Use a buffer solution

A solution that will experience little or no pH change

when small amount of base or acid are added to it

Here, pH will be kept at 5

 Other sample data
Absorbance/absorbance units
Trypsin concentration (%) 0s 15 s 30 s 45 s 60 s 75 s 90 s
1.0 1.97 1.38 0.68 0.22 0.11 0.09 0.06
0.8 1.97 1.39 0.75 0.23 0.13 0.11 0.09
0.6 1.97 1.42 0.87 0.41 0.14 0.10 0.06
0.4 1.97 1.57 1.21 0.84 0.47 0.30 0.11
0.2 1.97 1.78 1.51 1.28 1.02 0.82 0.58
0.0 1.80 1.80 1.80 1.80 1.80 1.80 1.80

Absorbance/absorbance units
Absorbance/absorbance units
pH 0s 15 s 30 s 45 s 60 s 75 s 90 s
Milk concentration (%) 0s 15 s 30 s 45 s 60 s 75 s 90 s
10 1.88 1.47 0.79 0.45 0.21 0.18 0.08
1.0 2.59 1.86 1.15 0.75 0.46 0.23 0.06
9 1.88 1.49 0.77 0.31 0.19 0.16 0.06
0.8 2.46 1.99 1.35 0.88 0.52 0.21 0.09
8 1.88 1.38 0.68 0.25 0.12 0.09 0.07
0.6 2.35 1.86 1.57 0.91 0.64 0.30 0.09
7 1.88 1.48 0.75 0.29 0.13 0.11 0.09
0.4 2.13 1.97 1.61 0.94 0.57 0.33 0.11
6 1.88 1.59 0.87 0.41 0.24 0.19 0.06
0.2 1.99 1.78 1.51 1.28 1.02 0.82 0.58
5 1.88 1.67 1.21 0.84 0.47 0.31 0.12
0.0 1.80 1.80 1.80 1.80 1.80 1.80 1.80
Answers to questions
1. Independent: depends on the investigation but appropriate answer from:
temperature, pH, trypsin concentration, substrate concentration
Dependent: rate of reaction in absorbance units s−1
2. Because the reaction is rapid and the milk (substrate) concentration quickly
declines. The rate slows as the substrate is used up. Therefore, it is only possible
to make valid comparisons at the start of the reaction, when controlled variables
such as substrate concentration are the same for all levels of the independent
variable.
3. A systematic error, because it would cause absorbance readings to be higher than
the true value for every measurement.
4. For the pH investigation: The rate of reaction of enzymes varies with pH, due to
changes in the shape of the active site. An enzyme has the highest rate of reaction
at its optimum pH. A buffer might be used to maintain pH at a suitable level.
For the temperature, substrate concentration and enzyme concentration
investigations. Temperature, because the rate of reaction of enzymes varies with
temperature. As temperature increases, particles gain more energy and more
collisions take place between enzyme and substrate particles. Enzymes have an
optimum temperature at which the rate of reaction is at its peak. Above that
temperature, enzymes will begin to denature, changing the shape of the active site
and preventing further catalysis. A water bath and thermometer could be used to
maintain a suitable temperature.

Answers to exam-style questions