BCE-lab report

H83 BCE LAB REPORT

NAME: Adnaan Malak

Student id: 012117

Group no: 7

Date of experiment: 30/03/2015

Date of submission: 06/04/2015

Lecturer: Dr. Lau Phei Li

SUMMARY: The following report is based on an experiment that was conducted to study how

enzyme catalysed reactions behave in different conditions. In this experiment

lipase was used as the enzyme that catalysed the hydrolysis of tributyrin into

butyric acid and glycerol. The Michaelis-Menten kinetics model was used as a

reference to study the effect of reaction rate on the concentration of substrate

and enzyme. The details on MM kinetics are further discussed in the report and

Hanes model is selected as a perfect model to calculate value of Kcat. Trend for

effect of change in substrate concentration and change in enzyme concentration

on the initial rate of reaction is also discussed in the discussion section of the

report.

INTRODUCTION: Enzymes are effective biological catalysts. They speed up the rate of reaction by

providing alternative route with lower activation energy. One of the classic

examples is the digestion /breakdown of fats found in the food to fatty acids in

the stomach by the enzyme lipase. Normally this reaction would take a long time

to occur and the food containing the necessary nutrients would just exit the body

but due to presence of lipase it is digested much faster. Enzymes are very

selective; they only bind with molecules which have desired active sites. Enzymes

get affected mostly by change in temperature or pH.

A study conducted has shown that enzymes generally follow the michaelis-

menton kinetics when they act as catalyst in the reactions. A much more detailed

abstract about the michaelis-menton was already discussed in the lab manual. In

our experiment, the enzyme tested is lipase and the substrate is tributyrin. Lipase

is mainly produced in the pancreas. The stomach also produces small amounts of

enzyme lipase. This enzyme catalyzes the hydrolysis of ingested food fat in the

body to fatty acids and glycerol so that it can be absorbed in the intestines.

Tributyrin is a triglyceride naturally found in foods such as butter and can be

described as a liquid fat with an acrid taste. The tributyic acid produced is

neutralized by NaOH to find the rate of reaction. The experiment was run until 5

minutes and a volume reading of NaOH and pH of the solution was noted every

30 seconds.

The main objectives of these experiments are:

• To determine the effect of the substrate concentration on the initial

rate of reaction for a constant concentration of enzyme.

• To determine Vmax and Km of the Michaelis-Menten kinetics.

• To determine the effect of enzyme concentration on the initial rate

of reaction.

• To determine Kcat

The hydrolysis of tributyrin to produce fatty acids and glycerol is assumed

to follow the enzymatic kinetics of the Michaelis-Menten model. This

model was developed in 1913. It provided a theoretical explanation for the

reaction rate using hypothesized reaction mechanism. It assumes that the

enzyme, E and substrate, S combine to form a complex ES, which then

dissociates into product P and free enzyme E.

S+E ↔ES→P+E

The Michaelis-Menten model is a pseudo steady state enzyme

kinetic. From this plot, it is difficult to determine the values of Km and

Vmax. So to determine the Vmax and the Km values, Lineweaver-Burk,

Eadie Hofstee and Hanes models were used. These models fairly give us a

good representation of Michaelis-Menten kinetics in a much more easy to

read plots, so that Vmax and Km values can be determined.

Lineweaver-Burk model:

[ ]

Y-axis is 1/Vo, Xaxis is 1/So

Slope equals to Km/Vm

Y-intercept equals to 1/Vm

Eadie Hofstee model:

Y-axis is Vo and x-axis is Vo/So

Slope equals to –Km

Y-intercept equals to Vmax

Hanes model:

Y-axis is So/Vo and x-axis is So

Slope equals to 1/Vmax

Y-intercept equals to Km/Vmax

RESULTS: EXPERIMENT 1: SUBSTRATE A

Volume of enzyme: 0.5ml

Table 1: Results of using 0.5ml enzyme and substrate A

Volume of enzyme:1.0ml


Time(min) pH Volume of NaOH added(ml)

0 6.81 2.064

0.5 6.81 0.093

1 6.99 0.146

1.5 7.02 0.170

2 7.02 0.214

2.5 7.02 0.214

3 7.02 0.234

3.5 7.02 0.254

4 7.02 0.274

4.5 6.99 0.292

5 7.01 0.304

Time pH Volume of NaOH added

0 6.83 2.035

0.5 6.52 0.186

1 6.82 0.286

1.5 7.01 0.342

2 6.99 0.378




0 6.82 2.042

0.5 6.6 0.096

1 6.8 0.176

1.5 7.01 0.234

2 7.01 0.260

2.5 7.00 0.280

3 7.01 0.290

3.5 7.01 0.310

4 7.01 0.324

4.5 7.01 0.340

5 7.02 0.356

2.5 6.99 0.422

3 7.03 0.446

3.5 7.01 0.460

4 6.99 0.478

4.5 7.03 0.498

5 7.02 0.510

Figure 1: Plot of volume of NaOH vs Time for substrate A

EXPERIMENT 2: SUBSTRATE B

Volume of enzyme:0.5mL

Table 4: Results of using 0.5ml enzyme and substrate B


0 6.81 3.322

0.5 6.47 0.160

1 6.62 0.286

1.5 6.72 0.388

2 6.76 0.478

2.5 6.82 0.564

3 6.87 0.644

3.5 6.91 0.722

4 6.97 0.798

4.5 6.99 0.860

5 7.01 0.918

y = 0.0529x + 0.0655

y = 0.0851x + 0.1515

y = 0.0615x + 0.0886

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 1 2 3 4 5 6

Vo

lum

e o

f N

aOH

ad

ded

(ml)

Time(min)

0.5 ml of enzyme

1 ml of enzyme

1.5 ml of enzyme

Linear (0.5 ml of enzyme )

Linear (1 ml of enzyme)


Volume of enzyme: 1.0ml

Table 5: Results of using 1.0ml enzyme and substrate B


0 6.89 2.874

0.5 6.41 0.224

1 6.61 0.292

1.5 6..77 0.378

2 6.92 0.456

2.5 7.01 0.504

3 7.01 0.540

3.5 7.01 0.574

4 7.0 0.606

4.5 7.0 0.632

5 7.01 0.658


Table 6: Results of using 1.5ml enzyme and substrate C


0 6.81 2.806

0.5 6.54 0.158

1 6.66 0.254

1.5 6.78 0.342

2 6.9 0.422

2.5 7 0.480

3 7 0.518

3.5 7 0.550

4 7.01 0.592

4.5 7 0.630

5 7.01 0.660

Figure 2: Plot of volume of NaOH vs Time for substrate B

EXPERIMENT 3: SUBSTRATE C




0 6.81 5.532

0.5 6.75 0.098

1 6.99 0.160

1.5 7.01 0.166

2 7.01 0.176

2.5 7.03 0.188

3 7.0 0.188

3.5 7.02 0.2

4 7.03 0.212

4.5 7.01 0.212

5 7.02 0.222

y = 0.1775x + 0.0853

y = 0.1153x + 0.154 y = 0.1221x + 0.1135 y = 0.1221x + 0.1135 y = 0.1221x + 0.1135 y = 0.1221x + 0.1135

0

0.2

0.4

0.6

0.8

1

1.2

0 1 2 3 4 5 6

Vo

lme

of

NaO

H a

dd

ed(m

l)

Time (min)

0.5 ml of enzyme

1 ml of enzyme

1.5 ml of enzyme


Linear (1 ml of enzyme )








0 6.81 5.498

0.5 6.55 0.128

1 6.77 0.218

1.5 6.97 0.290

2 7.01 0.310

2.5 7.01 0.320

3 7.00 0.328

3.5 7.00 0.336

4 7.00 0.346

4.5 7.01 0.354

5 7.01 0.366




0 6.8 5.37

0.5 6.68 0.126

1 6.94 0.208

1.5 7.02 0.226

2 7.03 0.236

2.5 7.00 0.233

3 7.01 0.246

3.5 7.03 0.258

4 7.01 0.258

4.5 7.02 0.268

5 7.03 0.278

Figure 3: Plot of NaOH vs Time for substrate C

EXPERIMENTAL VALUES FOR INTIAL RATE OF REACTION WITH VARIABLE

ENZYME CONCENTRATION:

Table 10 : Results of initial rate of reaction with variable enzyme concentration

Substrate A

So( mol/ml) Enzyme (LU/L) initial rate( mol/min)

1.14E-05 2.44E-02 2.65E-06

1.14E-05 4.76E-02 4.26E-06

1.14E-05 6.98E-02 3.08E-06

Substrate B


2.28E-05 2.44E-02 8.88E-06

2.28E-05 4.76E-02 5.68E-06

2.28E-05 6.98E-02 6.11E-06

y = 0.0328x + 0.0837

y = 0.0587x + 0.1256

y = 0.0397x + 0.1137

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 1 2 3 4 5 6

Vo

lum

e o

f N

aOH

ad

ded

Time (min)

0.5 ml of enzyme

1 ml of enzyme

1.5 ml of enzyme

Linear (1 ml of enzyme )



Substrate C


3.41E-05 2.44E-02 1.64E-06

3.41E-05 4.76E-02 2.94E-06

3.41E-05 6.98E-02 1.99E-06

Figure 4: Plot of enzyme concentration vs initial rate

LINEWEAVER-BURK MODEL DATA POINTS AND PLOT:

Table 11: Data for LINEWEAVER-BURK MODEL

0.00E+00

2.00E-06

4.00E-06

6.00E-06

8.00E-06

1.00E-05

0.00E+001.00E-022.00E-023.00E-024.00E-025.00E-026.00E-027.00E-028.00E-02

init

ial r

ate

(mo

l/m

in)

Enzyme concentration(LU/L)

Substrate A

Substrate B

Substrate C

LB Plot

0.5 ml v0 So 1/vo 1/So

A 2.65E-06 1.14E-05 3.78E+05 8.79E+04

B 8.88E-06 2.28E-05 1.13E+05 4.39E+04

C 1.64E-06 3.41E-05 6.10E+05 2.93E+04

1 ml v0 So 1/vo 1/So

A 4.3E-06 1.1E-05 2.4E+05 8.8E+04

B 5.7E-06 2.3E-05 1.8E+05 4.4E+04

C 2.9E-06 3.4E-05 3.4E+05 2.9E+04

Figure 5: Plot for LINEWEAVER-BURK model

Table 12: Values of Km and Vmax for LINEWEAVER-BURK MODEL

enzyme volume (ml) Km(mol/ml) Vmax(mol/min) Regression(R2) 0.5 -3.6E-06 2.2E-06 0.041

1 -3.6E-06 2.4E-06 0.072

1.5 -3.5E-06 3.2E-06 0.1554

y = -1.6478x + 455347

y = -1.0788x + 308592

y = -1.4967x + 411322

0.00E+00

1.00E+05

2.00E+05

3.00E+05

4.00E+05

5.00E+05

6.00E+05

7.00E+05

0.00E+00 2.00E+04 4.00E+04 6.00E+04 8.00E+04 1.00E+05

1/V

o(m

in/m

ol)

1/So(ml/mol)

0.5 ml enzyme concentration

1 ml enzyme concentration

1.5 ml concentration

Linear (0.5 ml enzymeconcentration )

Linear (1 ml enzymeconcentration )

Linear (1.5 ml concentration )

1.5 ml v0 So 1/vo 1/So

A 3.1E-06 1.1E-05 3.3E+05 8.8E+04

B 6.1E-06 2.3E-05 1.6E+05 4.4E+04

C 2.0E-06 3.4E-05 5.0E+05 2.9E+04

EDDIE-HOFSTEE MODEL DATA POINTS AND PLOT:

Table 13: Table for EDDIE-HOFTSEE MODEL

Figure 6 : Plot for EDDIE-HOFSTEE model

y = 2E-05x - 2E-07

y = 5E-06x + 3E-06 y = 1E-05x + 1E-06

0.00E+00

1.00E-06

2.00E-06

3.00E-06

4.00E-06

5.00E-06

6.00E-06

7.00E-06

8.00E-06

9.00E-06

1.00E-05

0.00E+00 1.00E-01 2.00E-01 3.00E-01 4.00E-01 5.00E-01

Vo

(mo

l/m

l)

Vo/So(ml/min)


1 ml enzyme concentration



Linear (1 ml enzymeconcentration )


Eadie Hofstee Plot

0.5 ml v0 So vo/[so]

A 2.65E-06 1.14E-05 2.32E-01

B 8.88E-06 2.28E-05 3.90E-01

C 1.64E-06 3.41E-05 4.80E-02

1 ml v0 So vo/[so]

A 4.26E-06 1.14E-05 3.74E-01

B 5.68E-06 2.28E-05 2.49E-01

C 2.94E-06 3.41E-05 8.60E-02

1.5 ml v0 So vo/[so]

A 3.08E-06 1.14E-05 2.70E-01

B 6.11E-06 2.28E-05 2.68E-01

C 1.99E-06 3.41E-05 5.82E-02

Table 14: Value of Km and Vmax for EDDIE-HOFSTEE model

HANES MODEL DATA POINTS AND PLOT:

Table 15 : Data for HANES model

3- Hanes plot

0.5 ml v0 So S0/vo

A 0.000002645 1.13772E-05 4.301386122

B 0.000008875 2.27543E-05 2.56386846

C 0.00000164 3.41315E-05 20.81188956

1 ml v0 So

A 0.000004255 1.13772E-05 2.673834616

B 0.000005675 2.27543E-05 4.009574024

C 0.000002935 3.41315E-05 11.62913079

1.5 ml v0 So

A 0.000003075 1.13772E-05 3.699891477

B 0.000006105 2.27543E-05 3.727163404

C 0.000001985 3.41315E-05 17.19470976

Table 16 : Values of Km and Vmax for HANES plot

enzyme concentration (ml) Km(mol/ml) Vmax (mol/min) Regression(R2)

0.5 -2.0E-05 -2.0E-07 0.8183

1 -5.0E-06 3.0E-06 0.3005

1.5 -1.0E-05 1.0E-06 0.4882

enzyme concentration km Vmax Regression

0.5 -1.00E-05 1.38E-06 0.6719

1 -7.24E-06 2.54E-06 0.7515

1.5 -8.92E-06 1.69E-06 0.859

Figure 7: Plot for HANES model

EFFECT OF VARIABLE SUBSTRATE CONCENTRATION ON THE INITIAL RATE

OF REACTION:

Table 17: Variable substrate concentration vs initial rate

y = 725598x - 7.2848

y = 393564x - 2.8511

y = 593066x - 5.2876

0

5

10

15

20

25

0 0.00001 0.00002 0.00003 0.00004

So/V

o

So


1ml enzyme concentration




Linear (1ml enzymeconcentration )

enzyme Concentration 0.024390244 0.04761905 0.069767442

Substrate concentration initial rate initial rate initial rate A 1.13772E-05 2.65E-06 4.26E-06 3.08E-06

B 2.27543E-05 8.88E-06 5.68E-06 6.11E-06

C 3.41315E-05 1.64E-06 2.94E-06 1.99E-06

Figure 8 : Plot for Variable substrate concentration vs initial rate

Table 18: Kcat values from HANES model

Enzyme Volume (ml) Kcat (mol/min)

0.5 0.00380

1.0 0.00358

1.5 0.00351

0.00E+00

2.00E-06

4.00E-06

6.00E-06

8.00E-06

1.00E-05

0 0.00001 0.00002 0.00003 0.00004

Init

ial R

ate

(mo

l/m

in)

Substrate Concentration (mol/ml)

enzyme concentration of0.02439

enzyme concentration of 0.048

enzyme concentration of0.0698

DISCUSSION:

EFFECT OF SUBSTRATE CONCENTRATION ON INITIAL RATE FOR CONSTANT

ENZYME CONCENTRATION

In theory more the substrate more the reaction rate should increase. This is

because more active sites are available a low substrate concentration. This

trend is clearly shown in Figure 8 .Whereas the substrate concentration

increases the reaction rate increases until it reaches a maximum point. In

our case for enzyme concentration of 0.02349,0.04761 and 0.06976 the

maximum values obtained for reaction rate are: 8.88E-06,6.11E-06 and

5.86E-06. When it reaches its maximum value then there is a drop in the

reaction rate as the enzyme gets saturated and thus there is no available

active sites for the substrate to bind with the enzyme.

According to (Jonathan crow, 2010) when the concentration of substrate

increases to a very high level, the substrate molecule competes for the

same active site on the enzyme to bind on. It can also be that the second

substrate binds on the specialized sites of enzymes where it can act as

allosteric inhibitor (Worthington-biochem). Hence due to these reason a

decrease in the reaction rate is observed .

DETERMING Vm and Kmax VALUE ASSUMING MICHAELIS-MENTEN

KINETICS

Values of Km and Vmax are determined by the three models: Lineweaver-

Burk, Eadie Hofstee and Hanes. Tables 12,14 and 16 displays the Km and

Vmax values. There are some limitations on using the three modified

models instead of the Michaelis-Menten original model. For Lineweaver-

Burk plot the linearity of LB plot is much less linear than the other two

plots; as also shown in figure 5,6 and 7 the R2 value for the LB plot is less

than the other two plots. There are several limitations associated with the

LB plot. These include:

• Difficult to determine how much graph paper is needed for the plot

to reach the x-axis.

• It allows low concentration of substrate to be used which can skew

the plot.

• Deviations from linearity are less easy to spot compared with other

means of interpretation.

For Eadie-Hofstee plot, the uneven spacing and the large values of inverse for

lower substrate concentration is overcome. But since Vo is used in both the axis

for plotting the graph the errors in measuring the Vmax are multiplied

considerably. From regression analysis shown in table 14 and 16 as compared to

Hanes plot has a lower R2 value which by no doubt makes Hanes plot the best

model to refers.

For hanes plot, the problem of uneven spacing and high values at low substrate

concentration is overcome by multiplying the term So to both the x-axis and the

y-axis (refer to introduction section to see the equation). But the disadvantage of

this model is that since So is use in both the axis. Due to which the drawback

results in an error value for true concentrations of but the overall error for the Km

and Vmax values are generally acceptable.

EFFECT OF ENZYME CONCENTRATION ON INITIAL RATE OF REACTION FOR

CONSTANT ENZYME CONCENTRATION

As it is observed from the figure 4 ; an increase in enzyme concentrations

leads to an increase in the rate of reactions for only substrate A and C . The

reason for substrate B not showing the similar trend can be because of the

systematic error or random error performed by Group number 22 from

which the values for 1.0 and 1.5 ml of enzyme added was taken. As for

substrate A and C the values for 0.5 and 1 ml were taken by my group

which shows the correct trend. This trend can be justified by the fact that

as the concentration of enzymes increases the availability of active sites

also increases which results in more substrates binding with the enzyme

resulting in a high rate of reaction. The initial rate of reaction h substrate A

is higher than C which could be because of substrate inhibition as explained

earlier.

Determining Kcat value for the enzyme

The Hanes plot is selected to determine the Kcat value because of the best

linear line obtained and has the largest R2 values.

How closely does the reaction comply with Michaelis-Menten kinetics?

Figure : ideal graph for MM equation

Figure: experimental graph obtained for MM equation

As it can be seen from the above two graphs for the following equation:

For the following experiment the reaction doesn’t completely comply with MM

equation. As it can also be seen from table 16 that the values obtained for Km are

in negative which suggests the involvement of experimental errors in our

experiments. But as said above it doesn’t completely comply which means that

the MM kinetics predicts that the Km and Kcat values do not change with the

increasing enzyme concentration. This trend is correctly followed by the Kcat

values that have been determined. But if we ignore the negative values then it

can be safely assumed that the reaction complies with the Michaelis-menten

Kinetics.

0.00E+00

2.00E-06

4.00E-06

6.00E-06

8.00E-06

1.00E-05

1.20E-05

1.40E-05

0 0.00001 0.00002 0.00003 0.00004

Vo

So

0.5 ml enzyme

1 ml enzyme

1.5 ml

CONCLUSION: The 5 criteria discussed in discussion part concludes the following:

The rate of reaction increases with increasing substrate concentration until

the enzyme active sites get saturated.

The rate of reaction increases with increasing enzyme concentration until

there is no more available substrate to be converted to products. Only

exception is for substrate B where experimental errors are involved.

The Vmax and Km values for all the LB, EH and Hanes model are

determined but only those from the Hanes model are selected.

The value of Kcat is determined using the Hanes model.

The reaction is not in perfect compliance to the Michaelis-Menten kinetic

model. However when the graph is plotted using the Hanes model, the

values of Vmax and Kcat can be determined to sufficient accuracy because

the lines obtained can be approximated to be linear with quite low errors.

REFERENCE:

1. John R. Holum, 1995. Elements of General, Organic and Biological Chemistry,

9th Edition. 9 Edition. Wiley.

2. Anon, (2015). [online] Available at: Mpbio.com, (2015). TRIBUTYRIN

(02103111) - MP Biomedicals. [online] Available at:

http://www.mpbio.com/product.php?pid=02103111&country=129 [Accessed

3 Mar. 2015].

3. Worthington-biochem.com, (2015). Enzyme Concentration (Introduction to

Enzymes). [online] Available at: http://www.worthington-

biochem.com/introbiochem/enzymeconc.html [Accessed 4 Mar. 2015].

4. Ucl.ac.uk, (2015). Untitled Document. [online] Available at:

http://www.ucl.ac.uk/~ucbcdab/enzass/substrate.htm [Accessed 27 Feb.

2015].

5. Worthington-biochem.com, (2015). Enzyme Concentration (Introduction to

Enzymes). [online] Available at: http://www.worthington-

biochem.com/introbiochem/enzymeconc.html [Accessed 1 Mar. 2015].

6. Dr Edward Group III DC, D. (2011). The Health Benefits of Lipase. [online]

Dr. Group's Natural Health & Organic Living Blog. Available at:

http://www.globalhealingcenter.com/natural-health/lipase/ [Accessed 3 Mar.

2015].

7. Abraham Mazur, 1971. Textbook of Biochemistry. 10th Revised edition

Edition. Saunders (W.B.) Co Ltd.

8. Enzymeessentials.com, (2015). Supplementing with Digestive Enzymes.

Lipase-Digestive enzyme to digest fats and lipids. [online] Available at:

http://www.enzymeessentials.com/HTML/lipase.html [Accessed 4 Mar.

2015].

9. Gaschott, T., Steinhilber, D., Milovic, V. and Stein, J. (2001). Tributyrin, a

Stable and Rapidly Absorbed Prodrug of Butyric Acid, Enhances

Antiproliferative Effects of Dihydroxycholecalciferol in Human Colon Cancer

Cells. The Journal of Nutrition, [online] 131(6), pp.1839-1843. Available at:

http://jn.nutrition.org/content/131/6/1839.full [Accessed 2 Mar. 2015].

10.Mpbio.com, (2015). TRIBUTYRIN (02103111) - MP Biomedicals. [online]

Available at:

http://www.mpbio.com/product.php?pid=02103111&country=129 [Accessed

4 Mar. 2015].

11.University of Maryland Medical Center, (2015). Lipase. [online] Available at:

http://umm.edu/health/medical/altmed/supplement/lipase [Accessed 6 Mar.

2015].

12.Athel Cornish-Bowden, 2012. Fundamentals of Enzyme Kinetics. 4 Edition.

Wiley-Blackwell.

13.2015. Untitled Document. [ONLINE] Available

at:http://www.ucl.ac.uk/~ucbcdab/enzass/substrate.htm. [Accessed 20

March 2015].

14.Jonathan Crowe, 2010. Chemistry for the Biosciences: The Essential

Concepts. 2 Edition. Oxford University Press.

APPENDIX:

SUBSTRATE CONCENTRATION

From Perry’s Chemical Handbook:

Density of Tributyrin, ρ= 1.032g/ml

Molecular weight of Tributyrin, MW= 302.36mg/mol

Initial Volume of Substrate, Vs= 1.0ml

Initial Total Volume, VT= 300ml

[ ]

Steps are repeated for Vs= 2.0ml and 3.0ml

REACTION RATE

SUBSTRATE A at 0.5ml enzyme:

(

) (

) (

)

(

) (

) (

)

Similar calculation is carried out for the other enzyme volumes of substrate A and

similarly for substrates B and C. All the calculated values are shown in table 10 .

DETERMINATION OF Km and Vmax values:

LINEWEAVER-BURK MODEL:

For figure 5 (enzyme volume 0.5ml) :

Slope =-1.6478

Intercept =455347

Equation:

[ ]


similarly for substrates B and C.

EADIE HOFSTEE MODEL:

For figure 6 (enzyme volume 0.5 ml):

Slope = 0.00002

Intercept = -2E-07

Equation:

[ ]

HANES MODEL:

For figure 7 (0.5 ml of enzyme):

Slope = 725598

Intercept =-7.2848

Equation: [ ]

[ ]


similarly for substrates B and C.

ENZYME CONCENTRATION

Volume of enzyme, VE

Initial concentration of enzyme,[Eo]

Total volume of solution (substrate + enzyme). VT

[ ]

Concentration of Enzyme, [E] = 24.390LU/m3

Similar calculation is carried out for enzyme volumes of VE=1.0ml and 1.5ml

DETERMINATION OF Kcat VALUES FROM HANES PLOT

Sample calculation for enzyme volume 0.5ml

[ ]

LAB RAW DATA:

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