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Report perfomed in the subject Biochemistry (L) in Politechnika Wroclawska

Report perfomed in the subject Biochemistry (L) in Politechnika Wroclawska

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INTRODUCTION

Enzymes are natural proteins that catalyses chemistry reaction of transformation of the substrate to Product. In the next

experiment the potato acidic enzyme will be used. This enzyme is be able to hydrolyse the substrate p-

nitrophenylphosphate and it transform in p-nitrophenol. This enzyme obeys the Michaelis Menten kinetic. It is mean that

the velocity equation is the next.

𝑽𝒎𝒂𝒙[𝑺]

𝒗𝒐 = that can be represented in the Michaelis Menten plot

𝑲𝒎

- 𝒗𝒎𝒂𝒙: Maximal rate of velocity, it is reached when all catalytic sites on the

enzyme are saturated with substrate.

- 𝑲𝒎: Constant of Michaelis Menten, it implies that half of the active sites

on the enzymes are filled. Specific to each different enzyme.

OBJETIVE

The main objective of this experiment is determinate the Michaelis constant and maximal velocity of reaction catalysed

of this enzyme Potato acidic phosphatase. Through spectrophotometry measurement of product will be determinate the

velocity of enzymatic reaction of hydrolysis. For this is necessary prepare a standard curve for p-nitrophenol.

MATERIALS

Reagents

- 0.5 M sodium hydroxide

- 5 mM solution of p-nitrophenylphoshate in 0.25 M acetate buffer pH 5.0

- Solution of potato acidic phosphatase in 0.25 M acetate buffer pH 5.0, CE o = 0,08 mg/ml

- Volumetric flask 10 ml

- Automatic pipettes and tips

- Cuvette

- Specol

- Thermostat 370C

1

PROTOCOL

1. Measurement of reaction velocity as concentration of substrate function.

- 21 dry tubes are tagged with the numbers 1, 2, 3..11 and 1’, 2’, 3’..11’.

- According to Table 1 solutions of 0.25M acetate buffer (pH=5.0) and 5.0mM p-nitrophenylphosphate are

added.

nr [ml] [ml] [ml]

1 1.75 0.05 0.10 2.0

1’ 1.85 0.05 - 2.0

2 1.70 0.10 0.10 2.0

2’ 1.80 0.10 - 2.0

3 1.65 0.15 0.10 2.0

3’ 1.75 0.15 - 2.0

4 1.60 0.20 0.10 2.0

4’ 1.70 0.20 - 2.0

5 1.50 0.30 0.10 2.0

5’ 1.60 0.30 - 2.0

6 1.35 0.45 0.10 2.0

6’ 1.45 0.45 - 2.0

7 1.20 0.60 0.10 2.0

7’ 1.30 0.60 - 2.0

8 1.05 0.75 0.10 2.0

8’ 1.15 0.75 - 2.0

9 0.80 1.00 0.10 2.0

9’ 0.90 1.00 - 2.0

10 0.50 1.30 0.10 2.0

10’ 0.60 1.30 - 2.0

11 1.80 0.00 0.10 2.0

- Then phosphatase is added to tubes without (‘) according to table 1 and samples are incubated 25 min in 370

- In the meantime, start second part of exercise “Standard curve”

- All tubes are incubated during 25 min. after this is added 2ml 0.5M solution NaOH, which stops reaction

catalysed by enzyme.

- Absorbance of solution is measured using wavelength 410nm, sample 11 is used like the blank sample.

2. Standard curve

o 200 l of p-nitrophenol are added to acetate buffer to final volume 10ml

o Dissolution is mixed by flask inversion

- Solution of p-nitrophenol diluted is transferred to a other test tube and It is tagged as P (product)

- 8 dry test tubes are tagged

2

- In the test tubes are adding solutions of 0.25 M acetate buffer pH = 5.0, 0.1 mM p-nitrophenol (in acetate

buffer) – standard (product) and 0.5 M NaOH in volume according to Table 2.

V NaOH [ml]

Sample nr V buffer [ml] V p-nitrophenol

[ml]

1 1.70 0.20 2.0

2 1.50 0.40 2.0

3 1.30 0.60 2.0

4 1.10 0.80 2.0

5 0.90 1.00 2.0

6 0.70 1.20 2.0

7 0.50 1.40 2.0

8 1.90 0.00 2.0

- Absorbance of each tube is measured in the spectrophotometer using wavelength 410nm, sample 8 is used like

the blank sample.

- The results are noted in the notebook and collected in the part of results.

3

CALCULATIONS AND DIS CUSSION OF RESULTS

1. Calculate concentration of p-nitrophenol after dilution – used standard in standard curve experiment 𝐶𝑝𝑘 [M].

1 0.2 5.1

2 0.4 10.3

3 0.6 15.4

4 0.8 20.5

5 1 25.6

6 1.2 30.8

7 1.4 35.9

Calculations performed:

0,20 𝑚𝐿 𝑜𝑓 𝑝𝑁𝑃𝐻 × × × × = 5,1 µ𝑀

1000 𝑚𝑙 3,9 𝑚𝐿 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 1𝐿 1𝑚𝑚𝑜𝑙

2. Prepare standard curve plot. And find out linear equation for this.

μmol

Sample Abs Calibration curve of p-nitrophenol

p-NPH

Blank 0 0 0.7

1 0.02 0.1 0.6

Absorbance

3 0.06 0.289 0.4

0.3

4 0.08 0.376 y = 4.6143x + 0.007

0.2

5 0.1 0.47 R² = 0.9997

0.1

6 0.12 0.56 0

7 0.14 0.649 0 0.05 0.1 0.15

p-nitrophenol (μmol)

Y = 4,6143x + 0,007

3. Calculate weight of phosphatase in reaction mixture 𝑚𝐸 [mg]. Volume of reaction mixture Vr = 1.90 ml.

The weight of phosphatase can be calculated knowing that their concentration in solution is 0,08 mg/mL.

Calculations performed:

0,08 𝑚𝑔 𝑜𝑓 𝐸𝑛𝑧𝑦𝑚𝑒

0,1 𝑚𝐿 𝑜𝑓 𝐸𝑛𝑧𝑦𝑚𝑒 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 × = 0,008 𝑚𝑔 𝑜𝑓 𝐸𝑛𝑧𝑦𝑚𝑒

1 𝑚𝐿 𝑜𝑓 𝐸𝑛𝑧𝑦𝑚𝑒 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

The concentration of the phosphatase in the reaction mixture can be obtained If we divide this amount into the final

volume of the reaction mixture, which is 1,9 mL.

Calculations performed:

0,008 𝑚𝑔 𝑜𝑓 𝐸𝑛𝑧𝑦𝑚𝑒

𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑝ℎ𝑜𝑠𝑓𝑎𝑡𝑎𝑠𝑒 𝑖𝑛 𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑚𝑖𝑥𝑡𝑢𝑟𝑒 = = 4,2 ∙ 10−3 𝑚𝑔/𝑚𝐿

1,9 𝑚𝐿 𝑜𝑓 𝑅𝑀𝑖𝑥

4

4. Calculate concentration of substrate (p-nitrophenylphosphate, p-NPH-P) ([S] calculated in μM) in reaction mixture

and inverse 1[𝑆].

solution (L/µmol)

1 0.05 131.6 0.0076

2 0.1 263.2 0.0038

3 0.15 394.7 0.0025

4 0.2 526.3 0.0019

5 0.3 789.5 0.0013

6 0.45 1184.2 0.0008

7 0.6 1578.9 0.0006

8 0.75 1973.7 0.0005

9 1 2631.6 0.0004

10 1.3 3421.1 0.0003

Calculations performed:

[𝑆] = 0,05 𝑚𝐿 𝑜𝑓 𝑠𝑜𝑙 𝑝𝑁𝑃𝐻𝑃 × × × × = 131, 6 µ𝑀

1000 𝑚𝑙 𝑜𝑓 𝑠𝑜𝑙 𝑝𝑁𝑃𝐻𝑃 1 𝑚𝑚𝑜𝑙 1,9 𝑚𝐿 1𝐿

Once you have the [p-NPH-P] you just have to make the inverse:

1 1 𝐿

= = 0,0076

[𝑆] 131,6 µ𝑚𝑜𝑙

5. Calculate absorbance ΔA410 /kor/:for amount of p-nitrophenol raised in enzymatic reaction. Using standard curve

from exercise A calculate amount of p-nitrophenol raised in enzymatic reaction (𝑛𝑝𝑡 calculated in µmol).

BLANK 0 0 0

1 0.369

0.367 0.078

1’ 0.002

2 0.585

0.580 0.124

2’ 0.005

3 0.692

0.685 0.147

3’ 0.007

4 0.716

0.701 0.151

4’ 0.015

5 0.856

0.842 0.182

5’ 0.014

6 0.905

0.885 0.191

6’ 0.020

7 1.003

0.977 0.211

7’ 0.026

8 1.092

1.059 0.229

8’ 0.033

9 1.129

1.072 0.232

9’ 0.057

10 1.138

1.084 0.235

10’ 0.054

Calculations performed:

5

∆𝐴𝑏𝑠 = 𝐴𝑏𝑠 𝑠𝑎𝑚𝑝𝑙𝑒 𝑋 − 𝐴𝑏𝑠 𝑠𝑎𝑚𝑝𝑙𝑒 𝑋′

The calibration curve performed with µmol of p-nitrophenol in “x” axis and absorbance in “y” axis is y = 4,6143x + 0,007

We have to isolate the “x” and substitute the “y” for the value of absorbance:

𝑦 − 0,007

𝑥=

4,6143

0,367 − 0,007

𝑥= = 0,078

4,6143

So, in sample 1, as the ΔAbsorbance is 0,367, the amount of p-nitrophenol is 0,078 µmol.

6. Calculate velocity of p-nitrophenol raising in enzymatic reaction 𝑣𝑐. Reaction time t = 25 min.

Vc

Sample

(µmol/min)

1 0.0031

2 0.0050

3 0.0059

4 0.0060

5 0.0073

6 0.0077

7 0.0085

8 0.0092

9 0.0093

10 0.0094

To calculate the velocity of the reaction we have to divide the amount of p-nitrophenol obtained into the time of

reaction, which is 25 minutes.

𝑛𝑝𝑡 (µ𝑚𝑜𝑙)

𝑣𝑐 =

𝑡 (𝑚𝑖𝑛)

0,078 µ𝑚𝑜𝑙

𝑣𝑐 = = 0,0031

25 𝑚𝑖𝑛

7. Calculate amount of product raised in 1 minute for 1 mg of enzyme – velocity 𝒗𝒔𝒑 and inverse.

1 0.389 2.571

2 0.622 1.607

3 0.737 1.356

4 0.755 1.325

5 0.909 1.100

6 0.956 1.046

7 1.057 0.946

8 1.147 0.872

9 1.161 0.861

10 1.174 0.852

6

We can calculate the 𝑣𝑠𝑝 by divide the 𝑣𝑐 value calculated previously into the mg of enzyme calculated in exercise 3:

𝑣𝑐 (µ𝑚𝑜𝑙/𝑚𝑖𝑛)

𝑣𝑠𝑝 =

𝑚𝐸 (𝑚𝑔)

0,0031 𝑣𝑐 (µ𝑚𝑜𝑙/𝑚𝑖𝑛)

𝑣𝑠𝑝 = =

0.008 𝑚𝐸 (𝑚𝑔)

(L/μmol) (µmol) (µmol/min) mgE·min) min/µmol)

Blank 0 0 0 0 0 0 0

1 0.367 131.6 0.0076 0.078 0.0031 0.389 2.571

2 0.580 263.2 0.0038 0.124 0.0050 0.622 1.607

3 0.685 394.7 0.0025 0.147 0.0059 0.737 1.356

4 0.701 526.3 0.0019 0.151 0.0060 0.755 1.325

5 0.842 789.5 0.0013 0.182 0.0073 0.909 1.100

6 0.885 1184.2 0.0008 0.191 0.0077 0.956 1.046

7 0.977 1578.9 0.0006 0.211 0.0085 1.057 0.946

8 1.059 1973.7 0.0005 0.229 0.0092 1.147 0.872

9 1.072 2631.6 0.0004 0.232 0.0093 1.161 0.861

10 1.084 3421.1 0.0003 0.235 0.0094 1.174 0.852

Mikaelis-Menten Kinetics

2.5

Vsp (µmol/ mg enz * min)

1.5

0.5

0

0 200 400 600 800 1000 1200 1400

[S] μM

7

b) 1/[S] in “x” axis against 1/Vsp in “y” axis.

R² = 0.9924

0,003

0,003

1/Vsp (mg enz*min/μmol)

0,002

0,002

0,001

-1/Km 0,001

1/Vmax

0,000

00,000 00,000 00,000 00,000 00,000 00,000 00,000 00,000 00,000

-0,001

1/[S] (L/μmol)

10. Using the curve calculate Michaelis Menten constant [μM] for tested enzyme and maximal celocity performed

enzymatic reactions:

𝑉𝑚𝑎𝑥 · [𝑆]

𝑉=

𝐾𝑀 + [𝑆]

1 𝐾𝑀 1 1

= · +

𝑉 𝑉𝑚𝑎𝑥 [𝑆] 𝑉𝑚𝑎𝑥

If we compare this equation with the structure of a line y = mx + n, the slope corresponds to Km/Vmax and the ordered

in origin is 1/Vmax.

With the graphical representation of the 1/[S] in the “x” axis and 1/V in the “y” axis we can obtain the equation of a line

and obtain the Km and Vmax from this one.

𝑦 = 231,24𝑥 + 0,7967

1

0,7967 =

𝑉𝑚𝑎𝑥

𝐾𝑀

231,24 =

𝑉𝑚𝑎𝑥

231,24 · 𝑉𝑚𝑎𝑥 = 𝐾𝑀

𝐾𝑀 = 1,25 · 231,24

𝑲𝑴 = 𝟐𝟗𝟎, 𝟐 µ𝑴

8

11. Summarize and discuss results.

With regard to the data of the concentration of p-nitrophenol calculated from the calibration curve we can say that is

quite accurate if we consider that the calibration curve has a regression coefficient very close to 1 (0,9999) which means

that all the points fit very good as a line.

When the [S] is been graphically represented against the velocity it can be appreciated a hyperbolic-like curve,

characteristic of Mikaelis-Menten enzymatic kinetics. The 4th value maybe should be higher to adjust better to the

hyperbolic curve, but it can be clearly appreciating the fast increase of the beginning of the curve and then a stabilization

when the enzyme it is supposed to be saturated, working at a Vmax velocity.

Thanks to the linearization of Lineweaver-Burk, the values of Vmax and Km can be easily obtained. The regression

coefficient of this line its also very close to 1 (0,9924) which means that the adjust to a line shape its quite good.

It’s been searched that values in the range between 10-8-10-6 M are considered that has a high affinity substrate-enzyme,

and a Km over 10-2 M are considered very low affinity1. The value that we have obtained its Km = 290,2 µM. If we convert

this value from µM to M it is 2,902 · 10-4 M, so their affinity is in the middle of the extreme values, neither too much

affinity not to low2.

The Vmax obtained show to us that 1 mg of enzyme its able to convert 1,25 μmol of subtract into product in one minute,

which is also quite good turnover number for an enzyme.

REFERENCES

1. Enzyme Kinetics. Available at:

https://www.chem.wisc.edu/deptfiles/genchem/netorial/modules/biomolecules/modules/enzymes/enzyme4.

htm. (Accessed: 24th November 2018)

https://employees.csbsju.edu/hjakubowski/classes/ch331/transkinetics/kmkcatvalues.htm. (Accessed: 24th

November 2018)

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