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. g L mol g L mol 01 . 0 1 002 . 0 09 . 108 0463 . 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. 2 2 1 1 V M V M 1 2 2 1 M V M V L mL L mM mL mM M V M V 08 . 1 1 1000 ) 3 . 46 ( ) 1 )( 05 . 0 ( 1 2 2 1 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. g L mol g L mol 1 . 0 1 01 . 0 6 . 182 055 . 0 of lysine - Calculate the volume of lysine stock solution needed to make a diluted lysine solution (20mM) using a 1 mL quartz cuvette. 2 2 1 1 V M V M 1 2 2 1 M V M V L mL L mM mL mM M V M V 6 . 363 1 1000 ) 55 ( ) 1 )( 20 ( 1 2 2 1 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)
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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.
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.
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
Cell3: Control- [pBQ]=0.01mM
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
UV/Vis Spectral Change of 0.025 mM of pBQ Control
Cell1: Control- [pBQ]=0.025mM
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
Cell1: Control- [pBQ]=0.025mM
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
UV/Vis Spectral Change of 0.05 mM of pBQ Control
Cell2: Control- [pBQ]=0.05mM
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
Cell2: Control- [pBQ]=0.05mM
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
UV/Vis Spectral Change of 0.075 mM of pBQ Control
Cell2: Control- [pBQ]=0.075mM
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
Cell2: Control- [pBQ]=0.075mM
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
UV/Vis Spectral Change of 0.01 mM of pBQ Control
Cell2: Control- [pBQ]=0.10mM
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
Cell2: Control- [pBQ]=0.10mM
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
Cell1: [pBQ]=0.01mM, [Lys]=20mM
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
Corrected Time (min)
Ab
sorb
ance
at
246n
m
File: K-MJK1-58-1
Cell1: [pBQ]=0.01mM, [Lys]=20mMHalf-life time (t1/2): 11.2min
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
Corrected Time (min)
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
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 7. Results of Reaction of [pBQ]=0.025mM, [Lys]=20mM Using UV/Vis Spectrometer
Time Elapsed UV/Vis Spectral Change Time-Dependent Absorbance Change at 246nm
Cell1: [pBQ]=0.025mM, [Lys]=20mM
0
0.5
1
1.5
2
2.5
200 300 400 500 600 700
Wavelength (nm)
Ab
sorb
ance
File: MJK1-64-1
Cell1: [pBQ]=0.025mM, [Lys]=20mM
0
0.2
0.4
0.6
0.8
1
1.2
0 50 100 150
Corrected Time (min)
Ab
sorb
ance
at
246n
m
File: K-MJK1-64-1
Half-life Time (t1/2): 10.8min Rate Constant (k): 0.0853min-1
Cell1: [pBQ]=0.025mM, [Lys]=20mMHalf-life time (t1/2): 10.8min
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
Corrected Time (min)
Ab
sorb
ance
at
246n
m
File: K-MJK1-64-1
Cell1: [pBQ]=0.025mM, [Lys]=20mMHalf-life time (t1/2): 10.8min
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
Corrected Time (min)
Ln
((A
f-A
t)/(
Af-
Ai)
)
File: K-MJK1-64-1
10
V-5. Reaction of 0.05 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
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 8. Results of Reaction of [pBQ]=0.05mM, [Lys]=20mM Using UV/Vis Spectrometer
Time Elapsed UV/Vis Spectral Change Time-Dependent Absorbance Change at 246nm
Cell1: [pBQ]=0.05mM, [Lys]=20mM
0
0.5
1
1.5
2
2.5
200 300 400 500 600 700
Wavelength (nm)
Ab
sorb
ance
File: MJK1-43-1
Cell1: [pBQ]=0.05mM, [Lys]=20mM
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 50 100 150
Corrected Time (min)
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
Cell1: [pBQ]=0.05mM, [Lys]=20mM
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
Corrected Time (min)
Ab
sorb
ance
at
246n
m
File: KK-MJK1-43-1
Cell1: [pBQ]=0.05mM, [Lys]=20mM
Half-life time (t1/2): 10.5min
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
Corrected Time (min)
Ln
((A
f-A
t)/(
Af-
Ai)
)
File: KK-MJK1-43-1
11
V-6. Reaction of 0.075 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
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 9. Results of Reaction of [pBQ]=0.075mM, [Lys]=20mM Using UV/Vis Spectrometer
Time Elapsed UV/Vis Spectral Change Time-Dependent Absorbance Change at 246nm
Cell1: [pBQ]=0.075mM, [Lys]=20mM
0
0.5
1
1.5
2
2.5
200 300 400 500 600 700
Wavelength (nm)
Ab
sorb
ance
File: MJK1-70-raw
Cell1: [pBQ]=0.075mM, [Lys]=20mM
0
0.20.4
0.6
0.81
1.2
1.41.6
1.8
0 50 100 150
Corrected Time (min)
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
Cell1: [pBQ]=0.075mM, [Lys]=20mM
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
Corrected Time (min)
Ab
sorb
ance
at
246n
m
File: K-MJK1-70-1
Cell1: [pBQ]=0.075mM, [Lys]=20mM
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
Corrected Time (min)
Ln
((A
f-A
t)/(
Af-
Ai)
)
File: K-MJK1-70-1
12
V-7. Reaction of 0.1 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
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 10. Results of Reaction of [pBQ]=0.1mM, [Lys]=20mM Using UV/Vis Spectrometer
Time Elapsed UV/Vis Spectral Change Time-Dependent Absorbance Change at 246nm
Cell1: [pBQ]=0.1mM, [Lys]=20mM
0
0.5
1
1.5
2
2.5
200 300 400 500 600 700
Wavelength (nm)
Ab
sorb
ance
File: MJK1-45-1
Cell1: [pBQ]=0.1mM, [Lys]=20mM
0
0.5
1
1.5
2
2.5
0 50 100 150
Corrected Time (min)
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
Cell1: [pBQ]=0.1mM, [Lys]=20mM
Half-life time (t1/2): 11.2min
y = -0.1131x + 2.5436R2 = 0.997
0
0.5
1
1.5
2
2.5
0 5 10 15
Corrected Time (min)
Ab
sorb
ance
at
246n
m
File: K-MJK1-45-1
Cell1: [pBQ]=0.1mM, [Lys]=20mM
Half-life time (t1/2): 11.2min
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
Corrected Time (min)
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
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.
Table 15. Results of Reaction of [pBQ]=0.01mM, [Lys]=10mM Using UV/Vis Spectrometer
Time Elapsed UV/Vis Spectral Change Time-Dependent Absorbance Change at 246nm
2. Reaction of 0.01 mM of pBQ and 30 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.
Table 16. Results of Reaction of [pBQ]=0.01mM, [Lys]=30mM Using UV/Vis Spectrometer
Time Elapsed UV/Vis Spectral Change Time-Dependent Absorbance Change at 246nm
3. Reaction of 0.01 mM of pBQ and 40 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 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.
Table 17. Results of Reaction of [pBQ]=0.01mM, [Lys]=40mM Using UV/Vis Spectrometer
Time Elapsed UV/Vis Spectral Change Time-Dependent Absorbance Change at 246nm
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
Time Elapsed UV/Vis Spectral Change Time-Dependent Absorbance Change at 246nm
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
Cell1: [pBQ]=0.01mM, [Ser]=20mMHalf-life time (t1/2): 20.2min
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
Corrected Time (min)
Ab
sorb
ance
at
246n
m
File: K-MJK1-93-pBQ0.01-Ser20-1
Cell1: [pBQ]=0.01mM, [Ser]=20mMHalf-life time (t1/2): 20.2min
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
Corrected Time (min)
Ln
((A
f-A
t)/(
Af-
Ai)
)
File: K-MJK1-93-pBQ0.01-Ser20-1
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
Time Elapsed UV/Vis Spectral Change Time-Dependent Absorbance Change at 246nm
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
Cell1: [pBQ]=0.01mM, [Thr]=20mMHalf-life time (t1/2): 16.8min
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
Corrected Time (min)
Ab
sorb
ance
at
246n
m
File: K-MJK1-97-pBQ0.01-Thr20-1
Cell1: [pBQ]=0.01mM, [Thr]=20mMHalf-life time (t1/2): 16.8min
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
Corrected Time (min)
Ln
((A
f-A
t)/(
Af-
Ai)
)
File: K-MJK1-97-pBQ0.01-Thr20-1
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
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)