Download Kinetics Cytochrome c Unfolding

Toward New Biology
Microdroplet Chemistry
Richard N. Zare, Stanford University, Stanford, California 94305 USA
[email protected]
Jae Kyoo Lee
Hong Gil Nam
Droplet-Droplet Fusion Kinetics
Kinetics Cytochrome c Unfolding
Cytochrome c is a single-domain, mainly helical
globular protein containing 104 amino acids and
one covalently attached heme group
Cytochrome c
Mass Spec
Protein
Unfolding
d
Charge
State
++
+
+ +++
+++ +
++++++
+++++
++
+
Acid
Unfolding
intermediate
Unfolded
Cytochrome c
SingleESI_CytochromeC_0.1%NH4OH_pH8.5 #22 RT: 0.18 AV: 1 NL: 3.69E6
T: FTMS + p ESI Full ms [200.00-4000.00]
Cytochrome c 100 µM
1545.80
100
+8
95
90
85
80
75
70
pH 8.5
Relative Abundance
65
+7
60
55
+9
50
45
40
1766.63
1374.16
35
30
+10
25
20
15
1822.05
1236.84
10
5
810.76
0
1030.95
954.03
800
1124.76
1149.14
1000
2060.73
1284.01
1200
1488.61
1428.59
1400
1730.60
1815.07
1587.82
1905.51 1979.70
1600
1800
2182.35 2249.88 2320.38
2000
2200
+10
m/z
SingleESI_CytochromeC_NoAcid #1 RT: 0.01 AV: 1 NL: 7.34E6
T: FTMS + p ESI Full ms [200.00-4000.00]
1374.27
100
+6
2472.67
2606.44
2400
2600
95
90
85
1236.85
80
75
pH 6.5
70
Relative Abundance
65
60
55
50
45
40
35
1545.80
30
25
20
1124.59
15
1098.95
1766.49
10
5
0
1561.04
748.25
1030.87
826.88 885.96
800
1189.46
1000
1289.67
1200
1452.15
1737.51
1781.47
1633.60
1400
1905.44
1600
1800
2060.74
2000
2178.27
2472.08 2537.78
2333.82
2200
2400
2600
m/z
SingleESI_CytochromeC_1%HCl_pH2 #325-343 RT: 2.80-2.96 AV: 19 NL: 2.84E5
T: FTMS + p ESI Full ms [200.00-4000.00]
1030.87
100
95
951.57
90
+12
1124.49
85
80
75
65
Relative Abundance
pH 2
1236.84
70
883.74
60
55
50
1374.16
45
1766.34
40
35
2060.73
30
1545.67
25
20
824.96
15
1737.97
1176.89
10
5
803.46
1334.25
1572.89
1462.91
1661.14
1996.45
1802.31 1904.06
2102.53
2266.18
2383.97
0
800
1000
1200
1400
1600
1800
2000
2200
2400
2521.44
Cytochrome c
unfolding for
different pH
values
Experimental Setup
Heated
Capillary
A
Mass
Spectrometer
B
A-B
Distance, x
Droplet
Generation
Mixing
Reaction
Desolvation
(Stopping of Reaction)
Speed of Droplet (m/s)
140
120
100
80
60
40
20
0
-1
0
1
2
3
4
5
6
7
8
x (mm)
Speed of liquid droplets as a function of the distance, x
between droplet fusion center to mass spectrometer inlet (red
line: linear fit for 0<x<0.8 mm, blue line: linear fit for 0.8<x<5
mm).
Estimated reaction time in liquid droplets calculated from the
measured droplet speeds. The inset shows the curvature for
0<x<1.2 mm.
Variation of Pure Water Droplet Size With Distance
Kinetics of Acid-Induced Cytochrome c Unfolding
𝐼 = 𝐴0 + 𝐴1 𝑒𝑥𝑝(−
•
•
•
•
𝑡
𝑡
) + 𝐴2 𝑒𝑥𝑝(− )
𝜏1
𝜏2
+9: t1 = 7.5 μs
+10: t1 = 30.0 μs, t2 = 25.6 μs
+11: t1 = 30.0 μs, t2 = 28.8 μs
+13: t1 = 49.0 μs
Test of Kinetics
The redox reaction between 2, 6 dichlorophenolindophenol
(DCIP) and ascorbic acid has been used widely to measure
reaction rates in the liquid phase:
Ascorbic
Acid
DICP
Dehydroascorbic
Acid
Leuco
Compound
Test of Kinetics
Droplets containing 100 μM ascorbic acid were fused with
droplets containing 1 μM DICP acid to provide pseudo-first
order kinetics based on the excess concentration of ascorbic
acid compared to DICP.
Test of Kinetics
Measured pseudo-first order rate constant is
1.0  0.2 x 105 s-1
Comparing Kinetics of Droplet Fusion to Bulk Solution
The pseudo-first order reaction rate of DCIP with excess
ascorbic acid in bulk solution is reported to be
116  3 s-1 (1) and 112  2 s-1 (2).
(1) Miao Z, Chen H, Liu P, Liu Y (2011) Development of submillisecond timeresolved mass spectrometry using desorption electrospray ionization. Anal Chem
83(11):3994–7.
(2) Karayannis MI (1976) Comparative kinetic study for rate constant determination
of the reaction of ascorbic acid with 2,6-dichlorophenolindophenol. Talanta
23(1):27–30.
CONCLUSION: Our measured rate of 1.0  0.2 x 105 s-1
is about three orders of magnitude higher than the rate
in bulk.
Jae Kyoo Lee, Samuel Kim, Hong Gil Nam, and Richard N. Zare "Microdroplet
fusion mass spectrometry for fast reaction kinetics" Proc. Natl. Acad. Sci. (USA)
112, 3898-3903 (2015).
Jae Kyoo Lee, Shibdas Banerjee, Hong Gil Nam, and Richard N. Zare,
"Acceleration of Reaction in Charged Microdroplets," Quarterly Reviews of
Biophysics 48, 437-444 (2015).
Pomeranz–Fritsch Reaction:
Acid-Promoted Synthesis of Isoquinoline
E. Schlittler and J. Müller, Helv. Chim. Acta. 1948; 31 (3); 914-924
Some Problems With Pomeranz-Fritsch Synthesis
J. M. Bobbitt and A. J. Bourque, Heterocycles 25, 601-616 (1987):
Unfortunately, yields are low and not always repeatable. Conc. H2SO4 tends
to destroy C, while dilute acids tend to hydrolyze its components.
Walter J. Gensler, “The Synthesis of Isoquinolines by the Pomeranz-Fritsch
Reaction,” Organic Reactions (2004):
Although a variety of methods has been reported for the cyclization step, all
involve the use of sulfuric acid. Sulfuric acid has been used alone, in
concentrations ranging from fuming acid to approximately 70% sulfuric acid.
A small deviation from the optimum acid concentration results in appreciable
decrease in the yield of isoquinoline.
Narrow Operating Window
Dr. Shibdas (Monon)
Banerjee
Experimental Setup
Pomeranz–Fritsch Reaction in Charged Microdroplets
+
3x10
8
2x10
8
1x10
8
[C+H]
m/z 222.1482
0
Counts
216 218 220 222 224 226 228
3x10
7
2x10
7
1x10
7
+
[D+H]
m/z 176.1064
0
168 171 174 177 180 183 186
1.0x10
6
5.0x10
5
+
[E+H]
m/z 130.0646
0.0
128
129
130
131
132
133
m/z
Data from 1 L/min MeOH solvent flow
but carried out also in ACN, H2O, and DMF
Jae Kyoo Lee, Shibdas Banerjee, Hong Gil Nam, and Richard N. Zare, QRB Discovery 48, 437-444 (2015).
Effect of Solvent Flow on Pomeranz–Fritsch Reaction in
Charged Microdroplet s
High flow
Fernandez de la Mora, J.;
Rossel- Liompart, J.
Proceedings of the 39th
ASMS Conference on Mass
Spectrometry and
Allied Topics; ASMS:
Nashville, TN, 1991; p. 441
Low flow
ES Capillary
21
10
18
(Iproduct/TIC)104
12
(IE / IC)*10
3
8
6
4
2
0
0.0
15
12
9
6
3
0.2
0.4
0.6
0.8
1.0
5
10
Solvent Flow (L/min)
15
20
25
30
0
0.0
0.2
0.4
0.6
0.8
1.0
5
Solvent Flow (L/min)
10
15
20
25
30
Variation of the Droplet Size With Flow Rate
𝑅∝
𝑅=
(𝑉𝑓)0.44
Initial Droplet
Radius (µL)
0.1
0.363 k
0.2
0.493 k
0.3
0.589 k
0.4
0.668 k
0.5
0.737 k
0.8
0.906 k
1
1.000 k
5
2.030 k
10
2.754 k
15
3.292 k
20
3.736 k
25
4.121 k
30
4.466 k
R = droplet radius
Vf = solvent flowrate
k = constant when a specific solvent is used
K. Tang and A. Gomez, J. Aerosol Sci.
Vol. 25, No. 6, pp 1237-1249 (1994).
12
10
(Iproduct/Ireactant)10
3
Solvent Flowrate
(µL/min)
𝑘(𝑉𝑓)0.44
8
6
4
2
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Droplet Radius (a.u.)
3.5
4.0
4.5
Effects of Ion Spray Voltage on Pomeranz–Fritsch Reaction in
Charged Microdroplets
3.0
(IE/IC) * 10
3
2.5
High Voltage
2.0
+
+
+
1.5
ES Capillary
+++
+
+
+
+ + +
+
1.0
+
+
+
+
+
Low Voltage
0.5
0.0
0
1
2
3
4
5
Spray Voltage (kV)
6
7
8
9
Reaction Rate Enhancement
The detected conversion to more than 1% isoquinoline in
droplet happened during 1 ms transit time.
Hours to days in bulk solution with 70% sulfuric acid
The reaction rate in the droplets has been
accelerated by a factor of a million or more.
“On Water” Reaction Rate Acceleration
D. C. Rideout and R. Breslow, JACS 102, 7817 (1980)
Cyclopentadiene Butenone
(12.1 mM
(0.4 mM)
and 25.5 mM)
Diels-Alder
reaction products
(endo + exo)
Quadricyclane
Solvent
neat
on H2O
Dimethyl
azodicarboxylate
Conc. [M]
4.53
4.53
Cycloaddition
product
T [C]
23
23
t [h]
48
0.17
Yield [%]
85
82
S. Narayan, J. Muldoon, M. G. Finn, V. V. Fokin, H. C. Kolb, K. B. Sharpless (2005)
Implications?
Poster Presented by Dr. Inho Nam
Accelerated Kinetics of Acid-Induced Chlorophyll
Demetallation in Microdroplets
Jae Kyoo Lee1, Hong Gil Nam2,, and Richard N. Zare1
1Department
of Chemistry, Stanford University, Stanford, California, 94305
USA.
2Center
for Plant Aging Research, Institute for Basic Science (IBS) and
Department of New Biology, DGIST, Daegu 711-873, Republic of Korea.
Chlorophyll in Photosynthesis
• Chlorophyll is the main photopigment used
to harvest light in photosynthesis by green
plants and cyanobacteria.
31
Chlorophyll a (no acid)
Chlorophyll a (with acid)
A stream of methanol microdroplets containing 40 μM
chlorophyll a or b collides with a stream of aqueous
microdroplets containing 35 mM hydrochloric acid (HCl;
pH = 1.46).
The rate of acid-induced chlorophyll demetallation was
about three orders of magnitude faster in the charged
microdroplet compared to that reported in bulk solution.
Kinetics of Chlorophyll Demetalation
The intensity of
chlorophyll a
decreases
whereas the
intensity of
phaeophytin a
increases as a
100
x = 0.6 mm
(t = 11 µs)
50
Relative abundance
• Mass spectra of
chlorophyll a at
different
travelling
distances of
fused
microdroplets.
871.57
893.54
PhaeophytinChlorophyll
a
a
0
100
x = 1.1 mm
(t = 17 µs)
50
0
100
x = 1.6 mm
(t = 23 µs)
50
0
100
x = 2.1 mm
(t = 29 µs)
50
0
860 870 880 890 900 910
m/z
34
Kinetics of Chlorophyll a
Demetallation
Normalized EIC (Chl a) / EIC (Chl a)0
The demetallation kinetics is first order
in [Chl a] and second-order in [H+]
d[Chl a]/ dt  k[Chl a][H ]2
1.0
[H+] >> [Chl a], so pseudo first order
kinetics
0.8
[Chl a]t  [Chl a]0 exp(kappt )
0.6
0.4
0.2
0.0
10
20
30
40
50
Measured rate constant:
k = 46 mM-2s-1 in in
microdroplet
K = 0.048 mM-2s-1 In bulk
solution
~ 1000 accelerations of
reaction
Time (s)
35
Light Stimulation on Chlorophyll
Demetallation Kinetics in
Microdroplets
LED Light
+
H
Reaction
Distance, x
Light Stimulation on Chlorophyll
893.54
[M+H+]+
915.52 [M+Na+]+
100
931.50 [M+K+]+
75
Relative Abundance
50
939.50 [M+O2+CH2+H+]+
25
0
939.50
[M+O2+CH2+H+]+
955.54
[M+2O2+CH2+H+]+
969.56
[M+2O2+2CH2+H+]+
985.54
[M+3O2+2CH2+H+]+
100
75
50
25
0
893.54
+]+
[M+H
915.52
[M+Na+]+
100
75
939.50 [M+O2+CH2+H+]+
50
25
0
840
• Chlorophyll
880
920
960
m/z
1000
1040
• Chlorophy
ll in Bulk
under
1800 PPF
(40
2)
mW/cm
• Chlorophy
Light
ll in for
20
min
Microdro
plets
under
Normalized EIC (Chl a) / EIC (Chl a)0
Effect of Light Stimulation on
Chlorophyll Demetallation Kinetics in
Microdroplets
Chlorophyll a without light
Chlorophyll a with light
1.0
0.8
0.6
0.4
0.2
0.0
10
20
30
40
Time (s)
• ~15% Enhanced Rate of Chlorophyll Demetallation under Light (1800 PPF) Stimu