i. sample calculations for p-benzoquinone (pbq) and amino ... · 1 min jung kang urp 2009 0601...

1

Min Jung Kang URP 2009 0601 Report 1 (pBQ)

I. Sample Calculations for p-Benzoquinone (pBQ) and Amino Acid (Lysine)

1. pBQ (FW= 108.09g/mol) - Calculate the concentration of pBQ stock solution with 0.01g of pBQ powder using a 2 mL

(=0.002L) volumetric flask.

gL

mol

g

L

mol01.0

1

002.009.1080463.0

of pBQ

- Calculate the volume of pBQ stock solution needed to make a diluted pBQ solution (0.05mM)

using a 1 mL quartz cuvette.

2211 VMVM 1

221 M

VMV

LmL

L

mM

mLmM

M

VMV

08.11

1000

)3.46(

)1)(05.0(

1

221

of pBQ stock solution (46.3 mM)

2. Amino acid: Lysine (FW=182.6 g/mol)

- Calculate the concentration of lysine stock solution with 0.1g of lysine powder using a 10 mL (=0.010 L) volumetric flask.

gL

mol

g

L

mol1.0

1

01.06.182055.0

of lysine

- Calculate the volume of lysine stock solution needed to make a diluted lysine solution (20mM)

using a 1 mL quartz cuvette.

2211 VMVM 1

221 M

VMV

LmL

L

mM

mLmM

M

VMV

6.3631

1000

)55(

)1)(20(

1

221

of lysine stock solution (55 mM)

M1 = Molarity of stock solution = 46.3 mM V1 = Volume of stock solution = X mL M2 = Molarity of diluted solution = 0.05 mM V2 = Volume of diluted solution = 1mL (cuvette)

M1 = Molarity of stock solution = 55 mM V1 = Volume of stock solution = X mL M2 = Molarity of diluted solution = 20 mM V2 = Volume of diluted solution = 1mL (cuvette)

2

Table 1. Volume of pBQ Stock Solution Needed to Make a Diluted pBQ Solution (1 mL cuvette)

Molarity of Diluted pBQ

solution (mM)

0.01

0.025

0.05

0.075

0.1

0.125

0.15

0.2

0.3

From 46.3 mM stock solution

(μL)

0.216

0.54

1.08

1.62

2.16

2.70

3.24

4.32

6.48

From 15.43 mM stock solution

(μL)

0.648

1.62

3.24

4.86

6.48

8.10

9.72

13.0

19.4

Table 2. Volume of Lysine Stock Solution Needed to Make a Diluted Lys Solution (1 mL cuvette)

Molarity of Diluted Lys

solution (mM)

20

50

From 55 mM stock solution

(μL)

363.6

909.1

Table 3. Chart for the Reaction of pBQ at Various Concentrations and Lysine at 20mM Using 1 mL Cuvette (Final volume is 1mL or 1000 μL)

Molarity of Diluted pBQ

solution (mM)

0.01

0.025

0.05

0.075

0.1

0.125

0.15

0.2

0.3

From 46.3 mM pBQ stock

solution (μL)

0.216

0.54

1.08

1.62

2.16

2.70

3.24

4.32

6.48

From 55mM Lys stock solution

(μL)

363.6

363.6

363.6

363.6

363.6

363.6

363.6

363.6

363.6

From 50mM Phosphate

buffer, pH7 (μL)

636.18

635.86

635.32

634.78

634.24

633.7

633.16

632.08

629.92

3

II. Instructions for Preparation of pBQ Stock Solution

1. Before transferring pBQ from the bottle to the balance, spray the weigh paper and spatula with the anti-static gun.

2. Weigh out approximately 10mg (0.01g) of pBQ powder to make 46.3 mM of pBQ stock solution

and transfer it into a 2 mL volumetric flask. 3. Rinse the mouth of the volumetric flask with 50mM phosphate buffer, pH 7, using a disposable

pipette. 4. Bring the solution to volume using the same phosphate buffer. 5. Place the cap on the flask and wrap the mouth of the flask with parafilm wax paper. 6. Shake well and place the volumetric flask in the sonicator for 10 minutes. 7. After 10 minutes has passed, remove the flask from the sonicator and place on ice until the

solution is needed. 8. Since pBQ is very reactive, fresh pBQ solution should be prepared.

III. Instructions for Preparation of pBQ and Amino Acid Samples Using Shimadzu UV/Vis

Spectrometer

1. Shimadzu UV/Vis spectrometer should be turned on and heated to 37°C at least 10 minutes before using it.

2. Perform autozero and baseline correction with the reference cell containing 50 mM phosphate

buffer, pH 7. 3. While sonicating pBQ stock solution for 10 minutes, prepare the quartz cuvettes with amino acid

and 50 mM phosphate buffer, pH 7. 4. Place the cuvettes in the sample holder of the spectrometer for 10 minutes to be equilibrated to

37°C before adding pBQ solution. 5. Prepare the timer and measure the time it takes to pipette aliquot of pBQ stock solution.

Measured time is going to be used when absorbance data is processed based on the corrected time. 6. Scan each cuvette. If each sample is triplicated, scan three samples at once since pBQ is less

reactive than ClpBQ. The reaction of ClpBQ is much quicker, so each cuvette should be scanned separately or individually.

4

IV. Kinetics Rule for pBQ and Amino Acid Reaction

1. If the samples are triplicated, scan three samples at once and correct the time.

2. Scan time interval is one fifth times of half-life time

2

15

1t .

3. Scan time interval for the pBQ control is 2 times of half-life time (2 x t1/2). 4. It takes approximately 1 minute to pipette pBQ solution into three cuvettes and place them in the

cell holder. It also takes 35 seconds for Shimadzu UV/Vis spectrometer to scan one cell. Thus, add 1 minute and 35 seconds (95sec) for the corrected time of the first cell, 1 minute and 70 seconds (130sec) for the second cell and 1 minute and 105 seconds (165 sec).

5. Half-life time (t1/2) is determined by selecting reactant λmax (=246nm) and first five data points.

Generate a plot of absorbance at 246nm vs. corrected time. Find the equation of line and calculate t1/2. Consider y-intercept as an initial absorbance. t1/2 is the time when half of initial absorbance (y-intercept) is being measured.

6. Scan the sample continually up to 4 times of half-life time (4 x t1/2) so that you have at least 5

data points to generate the rate constant. 7. A∞ can be detected when the sample is scanned at ten times of half-life time (10 x t1/2). 8. Use kinetic equation to find the rate constant.

ktAA

AA t

0

ln , where

9. Select the data generated within t1/2. Plot

0

lnAA

AA t vs. time. In other words, maximum range

of x-axis is one times of half-life time (1 x t1/2). 10. Find the equation of the line, and the slope is the rate constant.

*** Shimadzu UV/Vis Spectrometer Trouble Shooting ***

Perform autozero and baseline correction whenever software (UVProbe) is reopened. To delete UV/Vis spectra, perform right click on the spectra, select “Legend” and unmark

unnecessary spectra.

To delete unnecessary UV/Vis absorbance, perform right click on the absorbance table, select “Select All” and select “Hide” by performing right click.

A∞ : Absorbance at the time infinity At : Absorbance at time t A0 : Initial absorbance t : Reaction time k : Pseudo first order rate constant

5

V. Experimental Results and Discussion V-1. Standardization of pBQ Objective: To determine in which range of concentration of pBQ fits best to find out the rate

constant. Method: Initial absorbance at 246nm was recorded at different concentration of pBQ solution.

The samples were duplicated and the spectrum was scanned from 700 to 200nm by Shimadzu UV/Vis spectrometer. A plot of absorbance at 246nm vs. concentration of pBQ was generated to see the linear portion desirable to give the best accuracy and precision.

Table 4. Absorbance at 246nm measured by UV/Vis spectrometer at different concentrations of pBQ

Figure 1. Standardization of pBQ Using UV/Vis Spectrometer (File: MJK1-55-1)

Standardization of pBQ using UV/Vis spectrometer

0

0.5

1

1.5

2

2.5

3

3.5

0 0.1 0.2 0.3 0.4 0.5

[pBQ] (mM)

Ab

so

rba

nc

e a

t 2

46

nm

Conclusion: The concentration of pBQ should be selected in the range of less than 0.15mM of

pBQ to determine the rate constant of the reaction of pBQ with amino acid.

[pBQ](mM) Absorbance 0.01 0.025 0.05 0.075 0.1 0.125 0.15 0.2 0.25 0.35 0.4 0.5

246nm (cell1) 0.434 0.711 1.233 1.695 2.214 2.482 2.783 2.817 2.799 2.834 2.767 2.872

246nm (cell2) 0.363 0.692 1.2 1.702 2.094 2.408 2.696 2.767 2.767 2.767 2.834 2.799

Average 0.399 0.702 1.217 1.699 2.154 2.445 2.740 2.792 2.783 2.801 2.801 2.836

6

V-2. pBQ Control at different concentrations Objective: To determine the stability or absorbance change of pBQ in 50mM phosphate buffer,

pH7 in time dependent manner. Method: Each sample was triplicated and the spectrum was scanned from 700 to 200nm for 10

times of half-life (10 x t1/2) by Shimadzu UV/Vis spectrometer. Scan time interval was 2 times of half-life (2 x t1/2). A plot of absorbance at 246nm vs. time was generated. Only representative UV/Vis spectra were selected in this report.

Table 5. UV/Vis Spectral Change of pBQ Control at Different Concentrations

UV/Vis Spectral Change of 0.01 mM of pBQ Control

Cell3: Control- [pBQ]=0.01mM

0

0.05

0.1

0.15

0.2

0.25

200 300 400 500 600 700

Wavelength (nm)

Ab

sorb

ance

File: MJK1-72-1


0.222

0.224

0.226

0.228

0.23

0.232

0.234

0 50 100 150

Time (min)

Ab

sorb

ance

at

246n

m ∆A=0.233-0.223=0.01

File: MJK1-72-1



00.10.20.30.40.50.60.70.80.9

1

200 300 400 500 600 700

Wavelength (nm)

Ab

sorb

ance

File: MJK1-71-1-0.025


0.865

0.87

0.875

0.88

0.885

0.89

0.895

0.9

0 50 100 150

Time (min)

Ab

sorb

ance

at

246n

m

∆A=0.897-0.871=0.026

File: MJK1-71-0.025

7



0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

200 300 400 500 600 700

Wavelength (nm)

Ab

sorb

ance

File: K-MJK1-48-1


0.714

0.716

0.718

0.72

0.722

0.724

0.726

0.728

0.73

0.732

0 50 100

Time (min)

Ab

sorb

ance

at

246n

m

∆A=0.731-0.715= 0.016

File: K-MJK1-48-1



00.20.40.60.8

11.21.41.61.8

2

200 300 400 500 600 700

Wavelength (nm)

Ab

sorb

ance

File: MJK1-71-2-0.075


1.8151.82

1.8251.83

1.8351.84

1.8451.85

1.8551.86

1.8651.87

0 50 100 150

Time (min)

Ab

sorb

ance

at

246n

m

∆A=1.866-1.822= 0.044

File: MJK1-71-2-0.075



0

0.5

1

1.5

2

2.5

200 300 400 500 600 700

Wavelength (nm)

Ab

sorb

ance

File: K-MJK1-48-2


2.142.162.182.2

2.222.242.262.282.3

2.322.342.36

0 50 100 150

Time (min)

Ab

sorb

ance

at

246n

m

∆A=2.334-2.157=0.177

File: K-MJK1-48-2

8

V-3. Reaction of 0.01mM of pBQ and 20mM of Lysine Objective: To determine half-life time and the rate constant. Method: Each sample was triplicated and the spectrum was scanned from 700 to 200nm for 10

times of half-life (10 x t1/2) by Shimadzu UV/Vis spectrometer. Scan time interval was one fifth times of half-life (1/5 x t1/2). A plot of absorbance at 246nm vs. time was generated. Only representative UV/Vis spectra were selected in this report.

Table 6. Results of Reaction of [pBQ]=0.01mM, [Lys]=20mM Using UV/Vis Spectrometer

Time Elapsed UV/Vis Spectral Change Time-Dependent Absorbance Change at 246nm

Cell1: [pBQ]=0.01mM, [Lys]=20mM

00.2

0.40.60.8

11.21.41.6

1.82

200 300 400 500 600 700

Wavelength (nm)

Ab

sorb

ance

File: MJK1-58-1


0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 50 100 150

Corrected Time (min)

Ab

sorb

ance

at

246n

m

File: K-MJK1-58-1

Half-life Time (t1/2): 11.2min Rate Constant (k): 0.0892min-1

Cell1: [pBQ]=0.01mM, [Lys]=20mMHalf-life time (t1/2): 11.2min

y = -0.0143x + 0.3211

R2 = 0.9981

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 5 10 15


Ab

sorb

ance

at

246n

m

File: K-MJK1-58-1


y = -0.0892x + 0.2015

R2 = 0.9828

-1.2

-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0 5 10 15


Ln

((A

f-A

t)/(

Af-

Ai)

)

File: K-MJK1-58-1

9

V-4. Reaction of 0.025 mM of pBQ and 20mM of Lysine Objective: To determine half-life time and the rate constant. Method: Each sample was triplicated and the spectrum was scanned from 700 to 200nm for 10





0

0.5

1

1.5

2

2.5

200 300 400 500 600 700

Wavelength (nm)

Ab

sorb

ance

File: MJK1-64-1


0

0.2

0.4

0.6

0.8

1

1.2

0 50 100 150


Ab

sorb

ance

at

246n

m

File: K-MJK1-64-1

Half-life Time (t1/2): 10.8min Rate Constant (k): 0.0853min-1


y = -0.0566x + 1.2175

R2 = 0.9985

0

0.2

0.4

0.6

0.8

1

1.2

0 2 4 6 8 10 12


Ab

sorb

ance

at

246n

m

File: K-MJK1-64-1


y = -0.0853x + 0.1833

R2 = 0.983

-0.9-0.8-0.7-0.6-0.5

-0.4-0.3-0.2-0.1

00.1

0 2 4 6 8 10 12


Ln

((A

f-A

t)/(

Af-

Ai)

)

File: K-MJK1-64-1

10






0

0.5

1

1.5

2

2.5

200 300 400 500 600 700

Wavelength (nm)

Ab

sorb

ance

File: MJK1-43-1


0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 50 100 150


Ab

sorb

ance

at

246n

m

File: KK-MJK1-43-1

Half-life Time (t1/2): 10.5 min Rate Constant (k): 0.0809 min-1


Half-life time (t1/2): 10.5min

y = -0.0643x + 1.3541R2 = 0.9972

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 5 10 15


Ab

sorb

ance

at

246n

m

File: KK-MJK1-43-1



y = -0.0809x + 0.1661R2 = 0.984

-0.7

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0

0.1

0 5 10 15


Ln

((A

f-A

t)/(

Af-

Ai)

)

File: KK-MJK1-43-1

11






0

0.5

1

1.5

2

2.5

200 300 400 500 600 700

Wavelength (nm)

Ab

sorb

ance

File: MJK1-70-raw


0

0.20.4

0.6

0.81

1.2

1.41.6

1.8

0 50 100 150


Ab

sorb

ance

at

246n

m

File: K-MJK1-70-1

Half-life Time (t1/2): 10 min Rate Constant (k): 0.0966 min-1


Half-life time (t1/2): 10min

y = -0.0928x + 1.8489R2 = 0.998

0

0.2

0.40.6

0.8

1

1.21.4

1.6

1.8

0 5 10 15


Ab

sorb

ance

at

246n

m

File: K-MJK1-70-1


Half-life time (t1/2): 10min

y = -0.0966x + 0.1904R2 = 0.9917

-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0 2 4 6 8 10 12


Ln

((A

f-A

t)/(

Af-

Ai)

)

File: K-MJK1-70-1

12






0

0.5

1

1.5

2

2.5

200 300 400 500 600 700

Wavelength (nm)

Ab

sorb

ance

File: MJK1-45-1


0

0.5

1

1.5

2

2.5

0 50 100 150


Ab

sorb

ance

at

246n

m

File: K-MJK1-45-1

Half-life Time (t1/2): 11.2 min Rate Constant (k): 0.0853min-1



y = -0.1131x + 2.5436R2 = 0.997

0

0.5

1

1.5

2

2.5

0 5 10 15


Ab

sorb

ance

at

246n

m

File: K-MJK1-45-1



y = -0.0878x + 0.2103R2 = 0.9742

-1.2

-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0 5 10 15


Ln

((A

f-A

t)/(

Af-

Ai)

)

File: K-MJK1-45-1

13

Table 11. Average Rate Constant of pBQ at Different Concentrations and Lys at 20mM Avg. Rate Constant= 0.0915 min-1 Avg. Rate Constant= 0.1022 min-1

Rate Constants of pBQ and 20mM Lys (First data included)

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0 0.025 0.05 0.075 0.1 0.125

[pBQ] (mM)

Rat

e C

on

stan

t (m

in)-1

File: pBQ+Lys Table.xls

Rate Constants of pBQ and 20mM Lys(First data excluded)

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0 0.025 0.05 0.075 0.1 0.125

[pBQ] (mM)

Rat

e C

on

stan

t (m

in)-1

File: pBQ+Lys Table.xls

14

VI. Not Corrected Time vs. Corrected Time and First Data Included vs. First Data Excluded Objective: To determine how different rate constants and half-life are generated when not

corrected time frame and corrected time frame is used and when first data is included and excluded.

Table 12. Half-life and Rate Constants of pBQ and Lys When First Data is Included

pBQ+Lys First Data Included

Corrected Time Not Corrected Time

0.01mM+20mM Lys t1/2 (min) Rate Constant (min-1) t1/2 (min) Rate Constant (min-1)

Cell1 11.2 0.0892 10.4 0.0892 Cell2 11.2 0.0931 10.1 0.0833 Cell3 11.2 0.0987 9.8 0.0902

avg. 11.2 0.0937 10.1 0.08757 stdev. 2.2E-15 0.0048 0.3 0.00373


Cell1 10.8 0.0853 10 0.0853 Cell2 11 0.0859 9.9 0.0859 Cell3 10.9 0.0923 9.5 0.0923

avg. 10.9 0.0878 9.8 0.08783 stdev. 0.1 0.0039 0.2646 0.00388


Cell1 10.5 0.0809 10.2 0.0809 Cell2 9.6 0.0956 9.6 0.0956 Cell3 12 0.0709 10.2 0.0709

avg. 10.7 0.0825 10 0.08247 stdev. 1.21244 0.0124 0.3464 0.01242


Cell1 10 0.0966 9.2 0.0966 Cell2 10.6 0.0949 9.5 0.0949 Cell3 10.2 0.1048 8.8 0.1048

avg. 10.2667 0.0988 9.1667 0.09877 stdev. 0.30551 0.0053 0.3512 0.00529


Cell1 11.2 0.0878 10.5 0.0878 Cell2 10.8 0.097 9.7 0.097 Cell3 11 0.1002 9.6 0.1002

avg. 11 0.095 9.9333 0.095 stdev. 0.2 0.0064 0.4933 0.00644

15

Table 13. Half-life Time and Rate Constants of pBQ and Lys When First Data is Excluded

Conclusion: The corrected/not corrected time frame did not affect rate constants significantly.

pBQ+Lys First Data Excluded

Corrected Time Not Corrected Time


Cell1 11.1 0.0999 9.9 0.0999 Cell2 11.1 0.104 10 0.0952 Cell3 11.3 0.1085 10.3 0.1013

avg. 11.17 0.1041 10.1 0.0988 stdev. 0.11547 0.0043 0.208167 0.0032


Cell1 10.5 0.0954 9.8 0.0954 Cell2 10.8 0.0962 9.7 0.0962 Cell3 10.8 0.1018 9.5 0.1018

avg. 10.7 0.0978 9.667 0.0978 stdev. 0.173205 0.0035 0.152753 0.0035


Cell1 10.2 0.0904 10.5 0.0904 Cell2 9.6 0.1026 9.6 0.0904 Cell3 10.2 0.0925 12 0.0925

avg. 10 0.0952 10.7 0.0911 stdev. 0.34641 0.0065 1.212436 0.0012


Cell1 10.1 0.1044 9.3 0.1044 Cell2 10.9 0.1062 9.4 0.1062 Cell3 10.4 0.1114 9 0.1114

avg. 10.47 0.1073 9.23 0.1073 stdev. 0.404145 0.0036 0.208167 0.0036


Cell1 11 0.1005 10.2 0.1005 Cell2 10.8 0.1079 9.7 0.1079 Cell3 11 0.1116 9.6 0.1116

avg. 10.93 0.1067 9.83 0.1067 stdev. 0.11547 0.0057 0.321455 0.0057

16

Table 14. Chart for the Calculated Volume of Quinones at 0.01 to 0.075mM and Amino Acids at 10 to 40mM

Reactant Stock (mM) Final (mM) Vol. (μL)

1mL Cuvet Vol. (μL)

3mL Cuvet

pBQ (108.1g/mol) 0.004 g of pBQ in 2 mL vol.flask

0.010 0.54 1.62

18.50 mM

0.025 1.35 4.05

0.050 2.70 8.11

0.075 4.05 12.16

ClpBQ (142.54g/mol) 0.004 g of ClpBQ in 2 mL vol.flask

14.03 mM

0.010 0.71 2.14 0.025 1.78 5.35

0.050 3.56 10.69

0.075 5.35 16.04

MepBQ (122.12g/mol) 0.004 g of MepBQ in 2 mL vol.flask

16.38 mM

0.010 0.61 1.83

0.025 1.53 4.58

0.050 3.05 9.16

0.075 4.58 13.74

Lys (182.6g/mol) 0.1g of Lys in 10 mL vol.flask

54.76 mM

10 182.6 547.8 20 365.2 1095.6 30 547.8 1643.4 40 730.4 2191.2

Cys (121.2g/mol) 0.05 g of Cys in 10 mL vol.flask

41.25 mM

10 242.4 727.2 20 484.8 1454.4 30 727.2 2181.6 40 969.6 2908.8

Asp (133.1g/mol) 0.05 g of Asp in 10 mL vol.flask

37.57 mM

10 266.2 798.6 20 532.4 1597.2 30 798.6 2395.8 40 1064.8 3194.4

Ser (105.1g/mol) 0.05 g of Ser in 10 mL vol.flask

47.57 mM

10 210.2 630.6 20 420.4 1261.2 30 630.6 1891.8 40 840.8 2522.4

Thr (119.1g/mol) 0.05 g of Thr in 10 mL vol.flask

41.98 mM

10 238.2 714.6 20 476.4 1429.2 30 714.6 2143.8 40 952.8 2858.4

17

1. Reaction of 0.01 mM of pBQ and 10 mM of Lysine Objective: To determine t1/2 and the rate constant. Method: Each sample was triplicated and the spectrum was scanned continually from 700 to

200nm for 4 times of half-life (4 x t1/2) and A∞ was detected at 8 times of half life (8 x t1/2) by Shimadzu UV/Vis spectrometer. Scan time interval was 2 minutes and a plot of absorbance at 246nm vs. time was generated. Only representative UV/Vis spectra were selected in this report.




0

0.2

0.4

0.6

0.8

1

1.2

1.4

200 300 400 500 600 700

Wavelength (nm)

Ab

sorb

ance

File: MJK1-100-pBQ0.01-Lys10-raw

Cell1: [pBQ]=0.01mM, [Lys]=10mMt1/2: 20.4min

0

0.05

0.1

0.15

0.2

0.25

0.3

0 50 100 150 200


Ab

sorb

ance

at

246n

m

File: K-MJK1-100-pBQ0.01-Lys10-1

Avg. Half-life (t1/2): 19.6 min Avg. Rate Constant (k’): 0.0398 min-1


y = -0.0067x + 0.2733

R2 = 0.9989

0

0.05

0.1

0.15

0.2

0.25

0.3

0 2 4 6 8 10 12


Ab

sorb

ance

at

246n

m



y = -0.0379x + 0.0863

R2 = 0.9963

-0.8

-0.7

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0

0.1

0 5 10 15 20 25


Ln

((A

f-A

t)/(

Af-

Ai)

)


18


200nm for 4 times of half-life (4 x t1/2) and A∞ was detected at 8 times of half life (8 x t1/2) by Shimadzu UV/Vis spectrometer. Scan time interval was 2 minutes and a plot of absorbance at 246nm vs. time was generated. Only representative UV/Vis spectra were selected in this report.




0

0.5

1

1.5

2

2.5

200 300 400 500 600 700

Wavelength (nm)

Ab

sorb

ance

File: MJK1-100-pBQ0.01-Lys30-raw


0

0.05

0.1

0.15

0.2

0.25

0.3

0 20 40 60 80


Ab

sorb

ance

at

246n

m




y = -0.0173x + 0.2908

R2 = 0.9981

0

0.05

0.1

0.15

0.2

0.25

0.3

0 2 4 6 8 10 12


Ab

sorb

ance

at

246n

m



y = -0.1145x + 0.2186

R2 = 0.9917

-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0 2 4 6 8 10 12


Ln

((A

f-A

t)/(

Af-

Ai)

)


19


200nm for 4 times of half-life (4 x t1/2) and A∞ was detected at 8 times of half life (8 x t1/2) by Cary UV/Vis spectrometer. Scan time interval was 1.3 minutes and a plot of absorbance at 246nm vs. time was generated. Only representative UV/Vis spectra were selected in this report.




0

0.5

1

1.5

2

2.5

200 300 400 500 600 700

Wavelength (nm)

Ab

sorb

ance

File: MJK1-100-pBQ0.01-Lys40-Cell1

Cell1: [pBQ]=0.01mM, [Lys]=40mMt1/2: 5.7 min

0

0.05

0.1

0.15

0.2

0.25

0 10 20 30 40 50

Time (min)

Ab

sorb

ance

at

246n

m




y = -0.0201x + 0.231

R2 = 0.9948

0

0.05

0.1

0.15

0.2

0.25

0 2 4 6 8

Time (min)

Ab

sorb

ance

at

246n

m



y = -0.182x + 0.0394

R2 = 0.9926

-1.2

-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0 2 4 6 8

Time (min)

Ab

sorb

ance

at

246n

m


20

4. Reaction of 0.01 mM of pBQ and 20 mM of Serine Objective: To determine t1/2 and the rate constant. Method: Each sample was triplicated and the spectrum was scanned continually from 700 to

200nm for 4 times of half-life (4 x t1/2) and A∞ was detected at 8 times of half life (8 x t1/2) by Shimadzu UV/Vis spectrometer. Scan time interval was 3 minutes and a plot of absorbance at 246 nm vs. time was generated. Only representative UV/Vis spectra were selected in this report.

Table 17. Results of Reaction of [pBQ]=0.01mM, [Ser]=20mM Using UV/Vis Spectrometer


Cell1: [pBQ]=0.01mM, [Ser]=20mM

00.20.40.60.8

11.21.41.61.8

2

200 300 400 500 600 700

Wavelength (nm)

Ab

sorb

ance

File: MJK1-93-pBQ0.01-Ser20-raw

Cell1: [pBQ]=0.01mM, [Ser]=20mMHalf-life time (t1/2): 20.2min

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 50 100 150 200


Ab

sorb

ance

at

246n

m

File: K-MJK1-93-pBQ0.01-Ser20-1



y = -0.0095x + 0.3836

R2 = 0.9986

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 5 10 15


Ab

sorb

ance

at

246n

m



y = -0.0388x + 0.072

R2 = 0.9994

-0.8

-0.7

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0

0.1

0 5 10 15 20 25


Ln

((A

f-A

t)/(

Af-

Ai)

)


21

5. Reaction of 0.01 mM of pBQ and 20 mM of Threonine Objective: To determine t1/2 and the rate constant. Method: Each sample was triplicated and the spectrum was scanned continually from 700 to

200nm for 4 times of half-life (4 x t1/2) and A∞ was detected at 8 times of half life (8 x t1/2) by Shimadzu UV/Vis spectrometer. Scan time interval was 2.25 minutes and a plot of absorbance at 246 nm vs. time was generated. Only representative UV/Vis spectra were selected in this report.

Table 18. Results of Reaction of [pBQ]=0.01mM, [Thr]=20mM Using UV/Vis Spectrometer


Cell1: [pBQ]=0.01mM, [Thr]=20mM

00.20.40.60.8

11.21.41.61.8

2

200 300 400 500 600 700

Wavelength (nm)

Ab

sorb

ance

File: MJK1-97-pBQ0.01-Thr20-raw

Cell1: [pBQ]=0.01mM, [Thr]=20mMHalf-life time (t1/2): 16.8min

0

0.05

0.1

0.15

0.2

0.25

0 50 100 150


Ab

sorb

ance

at

246n

m

File: K-MJK1-97-pBQ0.01-Thr20-1



y = -0.007x + 0.235

R2 = 0.9991

0

0.05

0.1

0.15

0.2

0.25

0 2 4 6 8 10 12


Ab

sorb

ance

at

246n

m



y = -0.0494x + 0.095

R2 = 0.999

-0.9-0.8-0.7-0.6-0.5-0.4-0.3-0.2-0.1

00.1

0 5 10 15 20


Ln

((A

f-A

t)/(

Af-

Ai)

)


22

6. Reaction of 0.01 mM of pBQ and 20 mM of Aspartate Objective: To determine t1/2 and the rate constant. Method: Each sample was triplicated and continually scanned for 6 hours from 700 to 200nm.

After 24 hours, absorbance was obtained and A∞ was detected after 53 hours by Shimadzu UV/Vis spectrometer. Scan time interval was 600 sec (10 min) and a plot of absorbance at 246 nm vs. time was generated. Only representative UV/Vis spectra were selected in this report.

Table 19. Results of Reaction of [pBQ]=0.01mM, [Asp]=20mM Using UV/Vis Spectrometer

Time Elapsed UV/Vis Spectral Change Time-Dependent Absorbance Change at 246 nm

Cell1: [pBQ]=0.01mM, [Asp]=20mM

0

0.5

1

1.5

2

2.5

3

200 300 400 500 600 700

Wavelength (nm)

Ab

sorb

ance

File: MJK1-112-pBQ0.01-Asp20-raw

Cell1: [pBQ]=0.01mM, [Asp]=20mMt1/2: 228.4min

0

0.05

0.1

0.15

0.2

0.25

0 1000 2000 3000 4000

Time (min)

Ab

sorb

ance

at

246

nm

File: K-MJK1-112-pBQ0.01-Asp20-1

Avg. Half-life (t1/2): 230.5 min (3.8hr) Avg. Rate Constant (k’): 0.000257 min-1


y = -0.0005x + 0.2284R2 = 0.9952

0.205

0.21

0.215

0.22

0.225

0.23

0.235

0 10 20 30 40 50

Time (min)

Ab

sorb

ance

at

246

nm



y = -0.0026x - 0.004R2 = 0.9999

-0.7

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0

0 50 100 150 200 250

Time (min)

Ln

((A

f-A

t)/(

Af-

Ai)

)


23

7. Reaction of 0.01 mM of pBQ and 20 mM of Cysteine Objective: To determine t1/2 and the rate constant. Method: Each sample was triplicated and was continually scanned for 4 hours every 600 seconds

(10 min) from 700 to 200nm by Shimadzu UV/Vis spectrometer. Absorbance values were also obtained after 23.3 hours and 48.8 hours. These sample cuvettes were placed in the water bath at 37 ºC. A plot of absorbance at 299 nm vs. time was generated. Only representative UV/Vis spectra were selected in this report.

Table 20. Results of Reaction of [pBQ]=0.01mM, [Cys]=20mM Using UV/Vis Spectrometer

Time Elapsed UV/Vis Spectral Change Time-Dependent Absorbance Change at 299 nm

Cell1: [pBQ]=0.01mM, [Cys]=20mM

0

0.5

1

1.5

2

2.5

3

200 300 400 500 600 700

Wavelength (nm)

Ab

sorb

ance

File: MJK1-118-pBQ0.01-Cys20-1

Cell1: [pBQ]=0.01mM, [Cys]=20mM

0

0.02

0.04

0.06

0.08

0.1

0.12

0 20 40 60

Time (hr)

Ab

sorb

ance

at

299

nm

File: MJK1-118-pBQ0.01-Cys20-1

Data Analysis: After 23.3 hours have passed, the white particles were not appeared in the sample cuvettes but were shown when detected 48.8 hours after the sample has been prepared. It was not obvious to see the λmax and absorbance values at 299 nm were constantly increasing in time dependent manner, representing the product accumulation caused by adduct formation of pBQ with cysteine.

24

Table 21. Average t1/2 and Rate Constants of the Reactions of pBQ at 0.01 to 0.075mM and Lys at 20mM (File: pBQ + Lys Table)

0.01-0.075mM pBQ

+ 20 mM Lys

0.01mM pBQ 0.025mM pBQ 0.05mM pBQ 0.075mM pBQ

t1/2 (min)

k' (min-1)

t1/2 (min)

k' (min-1)

t1/2 (min)

k' (min-1)

t1/2 (min)

k' (min-1)

Cell1 11.2 0.0892 10.8 0.0853 10.5 0.0809 10 0.0966

Cell2 11.2 0.0931 11 0.0859 9.6 0.0956 10.6 0.0949

Cell3 11.2 0.0987 10.9 0.0923 12 0.0709 10.2 0.1048

Avg. 11.2 0.0937 10.9 0.0878 10.7 0.0825 10.2667 0.0988

Stdev. 0.0 0.0048 0.1 0.0039 1.21244 0.0124 0.30551 0.0053

Figure 1. Rate Constants vs. [pBQ] ([Lys]=20mM) (File: pBQ + Lys Table)

Rate Constants of pBQ and 20mM Lys

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0 0.025 0.05 0.075 0.1

[pBQ] (mM)

Rat

e C

on

stan

t (m

in)-1

Conclusion: The pseudo first order rate constants are independent of the concentration of pBQ.

25

Table 22. Average t1/2 and Rate Constants of the Reactions of ClpBQ 0.01mM and Lys at 10 to 40mM (File: pBQ + Lys Table)

0.01mM pBQ +

10-40mM Lys

10mM Lys 20mM Lys 30mM Lys 40mM Lys

t1/2 (min)

k' (min-1)

t1/2 (min)

k' (min-1)

t1/2 (min)

k' (min-1)

t1/2 (min)

k' (min-1)

Cell1 20.4 0.0379 11.2 0.0892 8.4 0.1145 5.7 0.182

Cell2 19.2 0.0403 11.2 0.0931 8.4 0.1228 5.5 0.1788

Cell3 19.2 0.0412 11.2 0.0987 8.4 0.1291 5.3 0.1859

Avg. 19.6 0.0398 11.2 0.0937 8.4 0.1221 5.5 0.1822

Stdev. 0.6928 0.0017 0.0 0.0048 0.0 0.0073 0.2 0.0036

Figure 2. Rate Constants vs. [Lys] ([pBQ]=0.01 mM) (File: pBQ + Lys Table)

Rate Constant vs. [Lys]([pBQ]=0.01mM)

0

0.05

0.1

0.15

0.2

0 10 20 30 40 50

[Lys] (mM)

Rat

e C

on

stan

t (m

in-1

)

Conclusion: The pseudo first order rate constants are dependent on the concentration of lysine.

26

Table 23. Average t1/2 and Rate Constants of the Reactions of 0.01mM pBQ and 20mM Amino Acids

0.01mM pBQ +

20 mM A.A

20mM Lys 20mM Ser 20mM Thr 20 mM Asp

t1/2 (min)

k' (min-1)

t1/2 (min)

k' (min-1)

t1/2 (min)

k' (min-1)

t1/2 (min)

k' (min-1)

Cell1 11.2 0.0892 20.2 0.0388 16.8 0.0494 228.4 0.0026

Cell2 11.2 0.0931 20 0.0397 16.7 0.0505 218.4 0.0026

Cell3 11.2 0.0987 20.4 0.0398 16.4 0.051 244.6 0.0025

Avg. 11.2 0.0937 20.2 0.0394 16.6 0.0503 230.5 0.00257

Stdev. 2.2E-15 0.0048 0.2 0.0006 0.2082 0.0008 13.22 5.77E-05

i. sample calculations for p-benzoquinone (pbq) and amino ... · 1 min jung kang urp 2009 0601...

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