ABS TRAC T

A COMPUTER INTERFACED RAPID SCANNING ABSORBANCE
AND FLUORESCENCE STOPPED-FIDW SYSTEM AND ITS
APPLICATION TO THE KINETICS 0F BIOIDGICAL REACTIONS

BY
GUAN-HUEI HO

A computer interfaced, double-beam, thermostated
stopped-flow system fOr fixed wavelength and rapid scanning
of'absorbance or light emission(fluorescence, luminescence
and reflectance) spectra has been constructed. The entire
system was constructed such that the solutions contact only
Pyrex, Teflon, Kel-F, polypropylene and quartz. A specially
designed all quartz, double 4-3et mixing and observation
cell(2 mm ID) with two optical path lengths has been made.
The syringes were made out of heavy-walled Pyrex precision
bore tubing(0.396" ID). Steel plungers with easily adjustable
Teflon cap seals were made to fit them. Two metal flags
mounted on the stOpping plunger provide timing pulses by
interrupting two light beams which are detected by separate
Imototransistors. The thermostat bath, which was made of
Plexiglas to provide good visibility, surrounds the flow
BYBtem and allows for temperature-dependent studies and for
establishing thermal equilibrium between the solutions and
the system. A rapid scanning monochromator is used to

disperse either the excitation or the emission beam. In the

GUAN-HUEI HO

absorbance mode, light beams are transmitted gig quartz
fiber Optics to both the sample cell and the reference cell,
and then to a pair of photomultipliers. The emitted light(or
reflected light), which is observed at 90° to the excitation
beam at the short path length, is conducted with a separate
quartz fiber bundle from the sample cell to the monochromator
and then to a photomultiplier. Sample and reference
photocurrents are converted to absorbance by use of a log
ratio operational amplifier, or into partially corrected
fluorescence by means of a ratio operational amplifier. The
absorbance or the fluorescence analog signal is sampled and
digitized at a nominal rate of 20.4 KHz, which is
synchronized to the rotation of the monochromator mirror
with the aid of a phase-locked-loop circuit. The signal is
transmitted to a remote PDT-B/I computer for data acquisition
and processing. The entire system was tested extensively by
studying several well-characterized fast reactions. Its
performance characteristics are excellent. The dead volume
was estimated to be 60‘pl for the short path length and
146‘pl for the long path length, which resulted in dead times
of 2.5 msec. and 6.5 msec., respectively, under a pushing
pressure ofvaO psig. This instrument is especially suited
to kinetics studies when only small volumes of the reagents
are available. At least 6 good pushes can be obtained from
only 5 ml of each reagent.

Scanning absorbance experiments on the redox reaction

of chromic acid and hydrogen peroxide in acidic solutions

GUAN-HUEI HO

were carried out at 22°C. The observed first-order rate
constants and the the calculated third-order rate constants
were computed and were fOund to be in good agreement with
published values.

The computer simulation of complex enzyme-catalyzed
reactions has been explored. Five hysteretic enzyme models
were investigated by numerically simulating the full time
course of the reaction progress curve. The Bates and Frieden
Model(D. B. Bates and C. J. Frieden, J. Biol. Chem., gig,
7878 (1973)) gives kinetically hysteretic behavior but does
not show allosteric behavior in the saturation of enzyme
activity. The Fit-Induced Model and the Monod—Wyman-Changeux
(MWC) Model(with n=2) both show kinetically hysteretic
response as well as allosteric saturation in the enzyme
activity. Slow conformational changes give hysteresis in the
time response of the reaction, while substrate binding at a
second site causes the allostery in the enzyme activity. The
cases with " zero cooperativity " and with " negative
cooperativity " for the MWC Model(with n=2) were also
simulated. The concept that an enzyme may have primitive
MichaelisAMenten activity together with conformational-
change-regulated MichaeliséMenten activity has been developed
and the dependence of the full time course of the reaction
on the initial substrate concentration and on the total
enzyme concentration was also numerically simulated.

The new instrument was first used to scan the kinetics

of firefly luciferase catalyzed reactions. Both the decay of

GUAN-HUEI HO

luciferin and the growth of enzyme-bound oxyluciferin occur
in at least two steps, each of which is first-order. A
transient intermediate absorption band was observed between
~420 nm and ~440 nm. A pseudo-isosbestic point was observed
at ~35} nm.

'The binding reactions of 1-anilino-8-naphthalene
sulfbnate and bovine serum albumin were scanned by measuring
the fluorescence changes. At least two steps are present in
the mechanism: an unobservably fast association process(the
quenching of BSA fluorescence) and a slower first-order
process(the enhancement of ANS fluorescence). Both processes
increase the fluorescence of ANS. The first-order process
shows dependence on the ratio of the equivalent
concentrations, (ANS)o/S(BSA)O, with a decrease in the first
order rate constant as the equivalent concentration ratio is
increased.

The first scanning fluorescence experiments with an
enzyme were done with a reaction catalyzed by lactate
dehydrogenase. The reaction was followed in both directions
by observing the changes in the NADH fluorescence.

The final scanning experiments were done with AMP
aminohydrolase catalyzed reactions by scanning the absorbance
from.~257 nm to N300 nm. The full time course of the
reactions fOr various concentrations of'5'-AMP at a fixed
AMP aminohydrolase concentration of 0.209 mg/ml and for
various total AMP aminohydrolase concentration at a fixed

S'-AMP concentration were determined. The dependence of the

GUAN-HUEI H0

full time course on various initial substrate and enzyme
concentrations shows characteristics similar to the
hysteretic enzyme model - MWC Model(with n=2). A set of

the kinetics and thermodynamic parameter values, which
approximately fit the experimental data, were found and the
simulated full time course behavior was compared with the

corresponding experimental data.

A COMPUTER-INTERFACED RAPID SCANNING ABSORBANCE
AND FLUORESCENCE STOPPED-FIOW SYSTEM AND ITS
APPLICATION TO THE KINETICS OF BIOIOGICAliREACTIONS

By

GUAN-HUEI HO

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements

fbr the degree of

DOCTOR OF PHIIOSOPHY

Department of Chemistry

1976

To

Shiu-Ying and My Parents

1‘

ACKNOWLEDGEMENTS

The author wishes to express his special gratitude to
Professor James L. Dye for his patience, guidance,
assistance and encouragement throughout the course of this
works I

He would also like to thank Professor C. H. Suelter
who served as my second reader for his valuble discussions,
helpful suggestions and criticisms.

The author also wishes to acknowledge the cooperation
of the MSU glass shop and the Chemistry Department machine
shop. Thanks go to Mr. M. Rabb for his help in the trouble
shooting of the interface system. Financial support from

the National Science Foundation is also acknowledged.

Last, I am grateful to my wife, Shiu-Ying, fbr her
patience and understanding which made accomplishment of

this goal possible.

111

 

Author and the Rapid Scanning Absorbance and Fluorescence
Stopped-Flow System. A - 1 kw Xenon lamp light source, B -
rapid scanning monochromator, 'C - photomultiplier tube, D -
storage oscilloscope, E - log ratio amplifier, F - mixing
and reference cells, G - thermostat.

1v

I.

II.

III.

TABLE OF CONTENTS

LIST OF TABLE 0 O I C O O O O C O O C C C O O 0
LIST OF FIGURE. 0 O O O O O O O O O O O O O O 0

PART ONE -- COMPUTER INTERFACED RAPID-SCANNING

ABSORBANCE AND FLUORESCENCE
STOPPED-Flow SYSTEM. . . . . . . . .

INTRODUCTION TO INSTRUMENTATION. . . . . . . . .

THE
AND

1.1--Rapid Scanning Spectroscopy . . . . . .

1.2--Considerations in the Instrumentation
and the Computerization of Stopped-
Flow System . . . . . . . . . . . . . .

1.3--Rapid Scanning Absorbance and
Fluorescence Stopped-Flow Apparatus . .

STOPPED-FLOW SYSTEM: DESIGN, CONSTRUCTION
TESTING. . . . . ... ._. . . . . . . . . . .
2.1--Flow System Design. . . . . . . . . . .
2.2--Mixing and Reference Cells. . . . . . .
2.3--Pushing and Stopping System . . . . . .
2.3.1--Syringe and Plunger Design . . .
2.3.2--Mechanical System and
Framework. . . . . . . . . . . .
2.3.3--Stopping Syringe System. . . . .
2.4--Solution Delivery System. . . . . . . .
2.5--Optical System Design . . . . . . . . .

THE COMPUTER INTERFACED SYSTEM: DATA ACQUISITION
AND PROCESSING O O O O O O O O O O C O O O O O O

3.1--Signal Averaging Scheme . . . . . . . .
3.2--Signal Enhancement in Real Time . . . .
3.3--Determination of Maximum Sampling Rate.
3.4--System Control and Timing . . . . . . .
3.5--System Hardware and Description . . . .
3.6--Remote Digital Transmission System. . .

V

Page
ix
xi

1O
1O
17
17

20
22
23
28

36
36
39
39
4O
42
44

3.7--Multiplication of Sampling Frequency.
3.8--System Software Description . . . . .
3.9--System Perfbrmance. . . . . . . . . .
3.10—-Calculation Schemes Used in Data
Analysis . . . . . . . . . . . . . .

IV. PERFORMANCE RESULTS. . . . . . . . . .4. . . .
' 4.1--0ptical Calibration Data. . . . . . .
4.2--Flow Calibration. . . . . . . . . . .
4.3--Mixing Cell Performance - Fixed
Wavelength Mode . . . . .'. . . . . .
4.3.1--Dead Time. . . . . . . . . . .
4.3.2--Mixing Efficiency and Stopping
Time . . . . . . . . . . . . .
4.3.3--Reliability of the System. . .
4.4--Quantitative Measurements - Scanning
Mode. . . . . . . . . . . . . . . . .
4.4.1--Scanning Absorbance. . . . . .
4.4.2--Scanning Fluorescence. . . . .
4.4.3--Scanning Reflectance . . . . .

PART TWO -- COMPUTER SIMULATION OF HYSTERETIC
ENZYME SYSTmS O O O C O O O O O O

V. HYSTERETIC ENZYMES . . . . . . . . . . . . . .
5.1--Hysteresis in Enzyme Systems. . . . .
5.1.1--Regulation of Enzyme Catalysis

5.1.2--The Enzyme Models Proposed . .

5.1.2.1--MWC Model . . . . . .

5.1.2.2--AKNF Model. . . . . .

5.1.2.3--HW Model. . . . . . .

5.1.2.4--BF Model. . . . . . .

5.2--Confbrmational Adaptibility and

Enzymatic Catalysis. . . . . . .

v1. SIMULATION OF HYSTERETIC ENZYME SYSTEMS. . . .
6.1--Computer Simulation 0 e e o e e e e e
6.2--Simulation of Hysteretic Enzyme

Page

45
45
49

49

51
51
57

59
59

61
63

\//

65 ”

65
71
79

85

85
85
85

89

92
92

93

95
95

VII.

VIII.

II.

System . . . . . . . . .
6.3--Hysteretic Enzyme Models
6.3.1-.M0del1 e e e e

6.3.2--Model 2 .
6.3.3--Model 3 .
6.3.4--Model 4 .
6.3.5--Model 5 .
6.4--Conclusions

PART THREE -- STOPPED-FLOW KINETICS

STUDIES

RAPID SCANNING ABSORBANCE STOPPED-FLOW KINETICS
STUDIES ON FIREFDY LUCIFERASE CATAEYZED REACTIONS
7.1--Introduction . . . . . . . . . . . . . .
7.2-4Materials and Experimental . . . .

7.3--Experimental Results and Preliminary

Data Analysis. 0 O O O O O O O O O O O O

7.4--Computer Fits of the Kinetics Data to

Combined First Order Mechanisms. . . . .
7.4.1--Consecutive First Order Kinetics.
7.4.2--Parallel First Order Kinetics . .

7eS--D18cu8810n e o e e e e e e e e e e e

RAPID SCANNING FLUORESCENCE STOPPED-FLOW
KINETICS STUDIES ON LACTATE DHIYDROGENASE

CATALYZED REACTIONS . . . . . . . . . . . . .
8.1--Intr0du0t10n . Q o o o o Q Q o o o o

8.2--Materials and Experimental . . . . . .
8.3--Experimental Results and Data Analysis

RAPID SCANNING FLUORESCENCE STOPPED-FIOW'KINETICS

STUDIES on BINDING or 1-ANILINO-8-NAPHTHALENE T0

vaNESERUMALBUMIN...............
901--Intr0duCt1°n e e e e e e e e e e e
9.2--Materials and Experimental . . . . . .

9.3-9Representative Kinetic Fluorescence

Spectra. . . . . . . . . . . . . . . .
9.4--Experimental Results and Data Analysis

vii

Page

99

99
119
136
150
168
179

181

181
181

184

185

189
192
193
197

203
203
204
205

212,
212
213

214
220

K.

XI.

XII.

RAPID SCANNING ABSORBANCE STOPPED-Flow KINETICS

STUDIES ON THE REDOX REACTION BETWEEN CHROMIC

ACID AND HYDROGEN PEROXIDE . . . . . . . . . . .
10.1--Introduction . . . . . . . . . . . . .
10.2--Materials and Experimental . . . . . .
10.3--Representative Kinetic Absorbance

Spectra and Time-cuts. . . . . . . . .

10.4--Experimental Results and Data Analysis

RAPID SCANNING ABSORBANCE STOPPED-FIOW'KINETICS

STUDIES OF AMP AMINOHYDROLASE CATALYZED

REACTIONS --- SUBSTRATE ACTIVATION . . . . . . .
11.1--Introduction . . . . . . . . . . . . .
11.2--Materials and Experimental . . . . . .
11.3--Experimental.Results and Data Analysis

SUGGESTIONS FOR FUTURE WORK. . . . . . . . . . .
APPENDIX1...................
APPENDIXZ......"..'...........
APPENDIX}...................
APPENDIX 4 . . . . . . . . . . . . . . . . . . .
APPENDIX 5 . . . . . . . . . . . . . . . . . . .
APPENDIX6...................
APPENDIX7...................
REFERENCES...................

viii

Page

229
229
230

230
234

243

~243

245
246

260
261
264
267
271
275
279
283
286

Table

2-2

6-1

6-2

6-3

9—1

10-1

IIST OF TABLES
Page

Scanning monochromator and phase-locked-loop
parameters. Output Sampling Frequency=20.4 KHz. 41

Flow time measurements. . . . . . . . . . . . . 61

Standard values of initial concentrations and

_ kinetic parameters fOr Model 1. . . . . . . . . 103

Standard values of initial concentrations of
substrate and enzyme, as well as kinetic
parameters, forMOdelz. . o o e e e e e e e 0123

Standard values of initial concentrations of
substrate and enzyme, and each kinetic
parameter, used in the simulation of Model 3. . 140

Standard values of initial concentrations of
enzyme and substrate, and each kinetic
parameter. fbr Model 4. . . . . . . . . . . . . 154

Standard values of the kinetic parameters, the
initial concentration of substrate and the
total enzyme concentration, fOr Model 5 . . . . 172

First order rate constants fer the luciferase
catalyzed reactions(at temperature 1890). . . . 196

First order rate constants for the luciferase
catalyzed reactions(at temperature 25°C). . . . 200

Observed first order rate constants(k b ) and
initial ANS fluorescence intensity(F 9,3

obtained from computer fitting, fOr %he binding
reaction of ANS to BSA in 0.1M phosphate pH 7.0
buffer solution at 22.0 1 0.100 . . . . . . . . 221

Observed pseudo-first order rate constants
(kob ) and calculated third-order rate
consgants(k), obtained from the computer
fitting, fer the reaction:
HCrO‘ + 2H20 + H*-——P Cr05 + EH20

6.1M ionic

(in strength at 22.0 x 0.100). . . . 238

ix

Figure

2-7

LIST OF FIGURES

Page

Schematic diagram of the thermostated stOpped-
flow apparatus. A-reactant reservoirs, B-joints
fOr reactants, C-electrically controlled 3-way
pneumatic valves, D-pushing syringes, E-mixing

and observation cell, F-stopping syringe, G-
reference cell, H-valves, I-pneumatic cylinder,
J-to vacuum and waste, K-quartz light fibers,
Ipthermostat bath, M-glass-to-plastic tubing
conneCtorS’ N-framemrko e e e e e e e e e e e e 11

Set-up fer the drilling of quartz capillaries.
A-Indexing head, B-From the Airbrasive unit,
C-Caplllarytube....o...........13

Schematic diagram of the mixing and
observation cell, A 42 cm, B 1.0 cm, 0 0.5
cm, E-cross-section of a mixer . . . . . . . . . 14

Set-up of cells and fiber Optics. A-Fiber
optics, B-Beam-splitter, C-Reference cell,
D‘MiXIJIg cell. 0 O O O O O O O O O O O O O O O O 18

Detailed view of a syringe used in the stopped-
flow system. A-Teflon cap, B~threaded rod,

C-Viton ”0” rings, D-fill position, E-rinse
position, F-locking nut. . . . . . . . . . . . . 19

Timing pulses from the flowalags. A-start flag
signal, B-flow velocity profile, C-stop flag
signal, D—locations of the metal flags on the
stopping plunger, E-locations of the
phototransistors, FgG-data.collection can be
initiated at one of these points, H-constant

flow velocity has been reached at this point . . 24

Timing diagram for a scanning experiment. F

and B refer to forward and backward scans

res ectively, t1 is called the time shift, t2

is efined as the flow time and t; is the ,
stoppingtimeeeeeeeeeeeoeeeeee.25

~Effective Xenon lamp spectrum showing the

relative PMT voltage obtained fOr both the

ferward and backward scans in a complete
revolution. Output of the stopped-flow

apparatus at a scanning speed of 75 spectra

per second; entrance slit=0.25 mm,PMT voltage
=500v, drum setting=1544, and nutation=0.64. . . 30

Absorbance circuit. A-fixed gain potentiometer,

Figure

2-10

2-11

2-12

2-13
2-14

2-15

2-16

2-17

2-18

2-19

B-high frequenc filter(controlled with
toggle switches . The amplifiers are from
Philbrick/Nexus Research and National
Semiconductor Corporation. . . . . . . . . .

Partially corrected fluorescence circuity.
A-fixed gain potentiometer, B-bias circuit.
All the amplifiers are from National
Semiconductor Corporation. . . . . . . . . .
Emsmple of the signal averaging scheme fOr

a fixed wavelength case. The numbers in
parentheses fOllowing the group number are
the number of samples averaged into each
point. The apprOpriate S/N values are

given in the triangles . . . . . . . . . . .

Computer interface for rapid scanning
absorbance and fluorescence stopped-flow
ayatem C O 0 O O O O O O O O O O O O O O O O

Flowbchart fOr the data acquisition routines

Block diagram of the computer interfaced
scanning stopped-flow system --- Absorbance

made 0 O O O O O O O O O O O O O O O O O O 0

Block diagram of the computer interfaced
scanning stopped-flow system ---
fluorescence mode. . . . . . . . . . . . . .

Holmium oxide glass spectrum collected with
the stopped-flow apparatus at a scanning
rate of 13.33 msec per spectrum. The
spectrum shown is the average of 100
individual spectra . . . . . . . . . . . . .

Permanganate(in 1M H SO solution) spectrum
collected with the egop ed-flow apparatus at
a scanning speed of 75 scans per second. The
spectrum shown is the average of 100
individual spectra . . . . . . . . . . . . .

Absorbance from the stopped-flow system 12
absorbance from a Cary 17 spectrometer for
aqueous solutions of’KMnO4 at a wavelength
Of 545 nm. 0 o 0 o 0 Q C I O O O O O O O I O
Absorbance from the stopped-flow system lg
absorbance from a Cary 17 spectrometer for
5'-AMP aqueous solution(ino0.0SM phosphate
pH 7.4 buffer solution, 22 C) at a

xi

Page

34

35

37

43 1/
47

52 \/‘

53 \j

54

55

56

Figure

2- 20

2-21

2-22

2-23

2- 24

2-25

2-26

2- 27

2- 28

Page
wavelengthof260nm............. 58

Time-development of the FeNCSZ+ complex at
456 nm obtained in a fixed wavelength
experiment. Total times for those portions
displayed are: A-1.03 sec, B-14 msec, C-

9 msec, D—5.8 msec. B and 0 show data
collection which started before the stop

,flag, D—shows data collection which started

at the time of the stop flag. . . . . . . . . 6O

Semilog plot of (A(to)-A(t))/(A(t )-A(tm))
vs time for the dead time computagion. Data

fitained from the decay of the reaction at

awavelengthof352nm...... ..... . 62

Time profile of the growth of the FeNCS2+

complex at 456 nm, used to estimate the

stepping time. The data obtained at 2 °C

with initial concentrations: 0.02M Fe *,

0.02M CNS“, in 0.2M HClO4 solution
reBPeCtively.................64

Overall spectral changes for the reaction of
0.01M Fe with 0.01M CNS" in 0.1M HClO

solution at 22°C. Spectra were collecteé

with the stOpped-flow system at a scanning

rate of13.33 msec per spectrum . . . . . . . 67

Time-cuts showing the forma ion of the FeNCS2+
ion from the reaction of Fe + with CNS" at

22°C in 0.1M H0104 solut on, with initial
concentrations: 0.01m Fe 1 and 0.01M CNS’ . . 68

Overall spectral changes for the reaction of
FeNCS "' and Ce(IV) in 0.7M HClO solution at
22°C. Spectra were collected wiIh the stopped
stopped-flow system at a scanning rate of

13.33 msec per spectrum . . . . . . . . . . . 69

Time courses at various wavelengths for the
reaction, PeNcS2+ + 4Ce(IV), in 0.7M HClO4

‘.801ut10nat22°Co..............7O

Fluorescence spectra of Fluorescein in

1 mM NaOH solution showing the concentration
sensitivity of this stopped-flow system. The
spectra were obtained at 22°C, slit=2.0 mm

and EMT-700V. The spectrum shown is the

average of 100 collected individual spectra . 72

Fluorescence spectra for 10’6M Fluorescein

xii

Figure

2-29

2-30

2-31

2-32

2-33

2-34

2-35

Page

in 1 mM NaOH solution showing the dependence

of fluorescence intensity on the opening of

slit. The spectra were obtained with this
stopped-flow system at 22°C and PMT power

supply 700V. Each spectrum shown is the

average of 100 collected individual spectra . 73

Fluorescence spectra fOr 10-7M Fluorescein

in 1 an NaOH solution showing the dependence

of fluorescence intensity on PMT power supply
voltage. The spectra were obtained with this
stopped-flow system at 22°C and slit=2.0. The
background has not been subtracted. Each

spectrum shown is the average of 100

collected individual spectra. . . . . . . . . 74

Fluorescence spectra for various

concentrations of NADH measured with this
stopped-flow system at 22°C in 0.05M

phosphate pH 7.4 buffer solution. Each

spectrum shown is the average of 100

collected individual spectra. . . . . . . . . 75

Fluorescence intensity 1g concentration fOr

NADH in 0.05M phosphate pH 7.4 buffer

solution at 22 C. The fluorescence

intensities were measured with this stopped—

flow system . . . . . . . . . . . . . . . . . 76

Fluorescence spectra for various

concentrations of Bovine Serum Albumin in

0.1M phosphate pH 7.0 buffer solution at

22°C. The spectra were obtained were this
stopped-flow system. Each spectrum Shown

is the average of 100 collected individual
apeCtraeeeeeeeeoeeeeeeeeee‘77

Fluorescence intensity vs concentration fbr
bovine serum albumin. TH? fluorescence
intensities of BSA in 0.1M phosphate pH 7.0
buffer solution were measured with this
stopped-flow system at 22°C . . . . . . . . . 78

Overall spectral changes fer the binding
reaction of DNA and P (II)(thiamine)(NH3)2

in 0.3M acetate pH 5.0 buffer solution at

22°C, with initial concentrations of 0.35

mg/ml DNA and 0.4 mg/ml Pt(II). The spectra

were obtained with a scanning rate of 13.33

msec per spectrum . . . . . . . . . . . . . . 80

Time course obtained from a fixed wavelength

xiii

Figure

2-36

2-37

5-1-1

5-1-2

5-1-3

5-1-4

6-1-1

push at 599 nm fOr the binding reaction of
DNA and Pt(II)(thiamine)(NH3)2 in 0.3M
acetate pH5.0 buffer solution at 22°C, with
initial concentrations: 0.175 mg/ml DNA and
0.4M/mlpt(II)...............

Time course obtained from fixed wavelength
pushes at 599 nm fOr the binding reaction of
DNA and Pt(II)(thiamine)(NH ) in 0.3M
acetate buffer solution at 328C, with
initial concentrations: A-O.35 /ml DNA

and 0.4 ms/ni PtéIIg, 3.0.175 mg ml DNA

and 0.4 mg/ml Pt II

Time course obtained from three successive
fixed wavelength puShes at 599 nm fOr the
binding reaction of DNA and Pt(II)(thiamine)
(NH3)2 in 0.3M acetate buffer solution at
220 , with initial concentrations: 0.175
mg/ml DNA and 0.4 mg/ml Pt(II). Note that
the decrease in overall reflectance was

due to deposition of the complex on the

cell Window. 0 O O O O O O O O O O O O O O O 0

Schematic representation of hyperbolic and
sigmoidal binding isotherms, the effect of
allosteric activators and inhibitors on the
sigmoidal isotherm and negative cooperativity
The ordinate is the fraction of sites
occupied, Y, although the initial velocity of
the enzymic reaction, v, also is commonly

used. 0 O O O O O O O O O O O O O O O O O O O

Allosteric models of'Monod, Wyman and
Changeux(MWC), and of Adair, Koshland,
Nemethy and Filmer(AKNF) for a fOur-subunit
enzyme. The squares and circles are different
confbrmations of the subunits, and S is the
subatrat e O O O O O O O O O O O O O O O O O 0

A general allosteric model(HN) fOr the

binding of substrate, S, to a fOur-subunit
enzyme. The squares and the circles are
different confOrmations of the subunits. The
MWC model is shown by dached lines and the
AKNF model by dotted lines. The free substrate
are omitted fOr the sake of clarity . . . . .

A hysteretic enzyme model proposed by Bates
and Frj'eden O O O O 0 O O O O O 0 O O O O O O

The BF Model, squares are less active enzyme

xiv

81

82

84

88

91

92

Figure

6-1-2a

6-1-2b
6-1-2c

6-1-3
6-1-4a

6-1-4b

6-1-5
6-1-6
6-1-7
6-1-8
6-1-9
6-1-10

6-1-11
6-1-12

6-1-13

ferns and circles are active enzyme fOrms,
S is substrate and P is product . . . . . . .

Simulated full time courses of product growth
and enzyme-substrate complexes, fOr Model 1 .

Simulated full time courses of product, each
species of emzyme and its complex at
$034200”, forMOdel1oeeeeeeeeee

Simulated full time courses of product growth
and free inactive enzyme at So=10500.pM,
fOr Model 1 . . . . . . .

Simulated time courses for various total
enzyme concentrations, fOr Model 1. . . . . .

Simulated time courses for various initial
substrate concentrations, fOr Model 1 . . . .

Simulated time courses of product growth fOr
various higher initial substrate
concentrations, fOr Model 1

Simulated time
various values

Simulated time
various values

courses of product(P) fOr
of'K1, fOr Model 1 . . . . . .

courses of product(P) fOr
°of K3, fOr Model 1 . . . . . .

Simulated time
various values

courses of product(P) for
or k5, for ”106181 1 e e e e e 0

Simulated time
various values

courses of product(P) fOr
of k2, fOr Model 1 . . . . . .

Simulated time
various values

Simulated time
various values

Simulated time courses of product growth rate
for various values of total enzyme
concentration, fOr Model 1. . . . . . . . . .

courses of product(P) fOr
Ofk69forMOdel1e e e e e 0

courses of product(P) fOr
of k-6, fOr Model 1. . . . . .

Simulated time courses of product growth
rate fOr various values of initial
concentration of substrate, fOr Model 1 . . .

Simulated time courses of product growth rate
fOr various values of kinetic parameters, K1,

XV

Page

100

103

104

104

106

106

107

109

109

110

110

111

111

113

113

Figure
6-1-14
6-1-15

6-1-16

6-1-17

6-1-18

6-2-1

6-2-2
6-2-3

6-2-4
6-2-5
6-2-6
6-2-7

6-2-8

K3,k23ndk4,f0rMOdel1..........

Simulated time courses of product growth
rate fOr various values of kinetic parameters
parameters, k5, k6, and k-6, fOr Model 1. . .

Simulated time courses of product growth fOr
gagiius total enzyme concentrations, fOr
o e 0 O O O O O O O O O O O O 0 O O O O 0

Simulated saturation curve of enzyme activity
at an enzyme concentration of 0.044 pM, fOr

Mode 1. For curve A, K =25,000‘pM and k2=7.5
sec’ , fOr curve B, K1=5000‘pM, k2=80 sec‘ .

Simulated saturation curve of enzyme activity
fOr an enzyme concentration of 0.044'pM, fOr
Model 1, with the parameter values of Frieden

Lineweaver-Burk plot, fOr Model 1. The
values of V0 and So were obtained from Curve
A of Figure 6-1-16. The Michaelis constant
and Vmax were fOund to be Km=2189i77‘pM and
Vhax=21.630.5‘pM/sec at (E)o=0.044‘pM,
respectively. . . . . . . . . . . . . . . . .

Fit-induced hysteretic enzyme model. The
enzyme has one effector site and one
substrate site. . . . . . . . . . . . . . . .

Fit-induced hysteretic enzyme model . . . . .

Simulated time courses of product growth and
intermediates, fOr Model 2. . . . . . . . . .

Simulated time courses of active fOrm enzyme-
effector complex fOr various total enzyme
concentrations, fOr Model 2 . . . . . . . . .

Simulated time courses of enzyme-effector-
substrate complex fOr various total enzyme
concentrations, fOr Model 2 . . . . . . . . .

- Simulated time courses of product growth fOr

various total enzyme concentrations, fOr
"Odel 2 O O O O I O O O O O O O C O O O O O 0

Simulated time courses of product growth
fOr various initial substrate concentrations,
forMOdQIZOeeeeeeeeeeee....

Simulated time courses of product growth fOr

xvi

Page
114

115

116

116

117

118

120
122

123

125

125

126

126

Figure

6-2-9

6-2-10
6-2-11
8-2-12
6-2-13
6-2-14

6-2-15
6-2-16
6-2-17
6-2-18
6-2-19

6- 2-20

6-5-1

6-5-2

' fOr Model 2 .

. Model 2 . . .

various values of K1, for Model 2 . . . . . .

Simulated time
various values

courses of product growth fOr
Of K3, for MOdel 2 e e e e e 0

Simulated time
various values

courses of product growth fOr
Of k2, for MOdel 2 e e e e e 0

Simulated time
various values

courses of product growth fOr
Of K4, for MOdGl 2 e e e e e 0

Simulated time
various values

courses of product growth fOr
of k-6, fOr Model 2. . . . . .

Simulated time
various values

courses of product growth for
of kg, for Model 2 . . . . . .

Simulated time
various values

Simulated time courses of product growth for
various total enzyme concentrations, fOr
Model 2, using Eb-time) as time scale. . . .

courses of product growth fOr
0fk5,f01‘M0d912. e e e e 0

Simulated time courses of product growth rate
fibr various total enzyme concentrations, fOr
odel 2 .

Simulated time courses of product growth rate
fOr various initial substrate concentrations,

Simulated time courses of product growth rate
for various values of K1, k2, K3 and K4, for
Model 2 . .

Simulated time courses of product growth rate
fOr {arious values of k5, kg and k-5, fOr
Mode 2 . .

O O O O O O O O O O O O O I O O 0

Simulated saturation curve of enzyme activity'

at an enzyme concentration of 0.044 uM, fOr

Compound hysteretic enzyme model fOr one
subunit with one effector site and one
catalytic site. Circle is catalytic site
and half circle is effector site. . . . . . .
Compound hysteretic enzyme model fOr one
subunit with one effector site and one
catalytic site. . . . . . . . . . . .

xvii

Page
128

128

129

129

130

130

131

131

132

132

133

134

135

157

138

Figure Page

6-3-3a Simulated time courses of product growth,
each fbrm of free enzymes, and the i
intermediates, fOr Model 3 . . . . . . . . . . 140

6-3-3b Simulated time courses of product growth,
each fOrm of free enzyme and its complex
With K4=K7 and kszkag for made]. 30 e e e e e o 141

6-3-3c Simulated time courses of product growth and
: inactive form free enzyme with K4=K7 and
k5=k8 at S°=Z1OOPM’ for "Odel 3 o e e e e o e 141

6-3-4 Simulated time courses of product growth fOr
various total enzyme concentrations, fOr
"Odel 3. O O O O O O O O O O O O O O O 0 O O O 143

6-3-5 Simulated time courses of product growth fOr
various initial substrate concentrations,
fOI‘MOdel3..................143

6-3-4b Simulated time courses of product growth fOr
various total enzyme concentrations with
K4=K7 and k5=k8’ for made]. 3 o o o o o o o o o 144

6-3-5b Simulated time courses of product growth fOr
various initial substrate concentrations
With K4=K7 and ICE-aka, for M0del 30 e e e e e e 144

6-3-6 Simulated time courses of product growth fOr
various values of K1, fOr Model 3. . . . . . . 145

6-3-7 Simulated time courses of product growth fOr
various values of K3, fOr Model 3. . . . . . . 145

6-3-8 Simulated time courses of product growth for
various values of kg, for Model 3. . . . . . . 146

6-3-9 Simulated time courses of product growth fOr
various values of k6, fOr Model 3. . . . . . . 146

6-3-10 Simulated time courses of product growth fOr
various values of K4, fOr Model 3. . . . . . . 147

6-3-11" Simulated time courses of product growth fOr
various values of K7, fOr Model 3. . . . . . . 147

6-3-12 Simulated time courses of product growth fOr
various values of k5, fOr Model 3. . . . . . . 148

6-3—13 Simulated time courses of product growth fOr
various values of k8, fOr Model 3. . . . . . . 148

xviii

Figure
6—3—14

6-5-15

6-4-1_

6-4-2

6-4-3a

6-4-3b

6-4-3c

6-4-4a

6-4-5a

6-4-4b

6-4-5b

6-4-4c

5-4-6

Page
Simulated time courses of product growth for
various values of k-6, fOr Model 3 . . . . , , 149

Simulated saturation curve of enzyme activity
fOr an enzyme concentration of 0.044,pM, fOr
made]. 3. O O O O O O O O O O O O O O I O O O O 149

Two subunit, symmetric and concerted,

.hysteretic enzyme model. . . . . . . . . . . . 151

Two subunit, symmetric and concerted,
hysteretic enzyme model. This is the case of
‘MWC model with n=2 . . . . . . . . . . . . . . 153

Simulated full time courses of product(P)
growth, inactive fOrm free enzyme(E) and active
fOrm enzyme-substrate(E§2), fOr Model 4. . . . 154

Simulated full time courses of product growth,
free enzymes and their complexes with k9=k11,
for Model 4. O O O O O O O O O O O O O O O O O 155

Simulated full time courses of product growth,
inactive fOrm enzyme(E) and enzyme complex(E§2),
with k93k11at So=1260‘mM, fOr Model 4. . . . . 155

Simulated time courses of product growth fOr
various total enzyme concentrations, for
Madel 4e 0 e e e e e o e e e e e e e e e e e o 157

Simulated time courses of product growth fOr
various initial substrate concentrations, fOr
MOdel 4. O O O O O O O O O O O O O O O O O O O 157

Simulated time courses of product growth fOr
various total enzyme concentrations with
k9=k11g fOr MOdel 4e 0 e e e e e e e e e o e e 158

Simulated time courses of product growth fOr
various initial substrate concentrations with
k9=k11, fbr Medal 4. e e e e e e e o e e e e e 158

5 Simulated time courses of product growth fOr

various total enzyme concentrations with
k k11, fOr Model 4, using (Eb~time) as
t me Scale 0 O O O O O O O O O O O O O O O O O 159

Simulated time courses of product growth fOr
various values of K1, for Model 4. . . . . . . 161

Figure
6-4-7
6-4-8
6-4-9 :

6-4-10

6-4-11a

6-4-11b

6-4-12a

5-4-12b

6-4-13

6-5-1

6-5-2
6-5-3

Page

Simulated time courses of product growth for
various values of‘K5, fOr Model 4 . . . . . . . 161

Simulated time courses of product growth fOr
various values of kg, for Model 4 . . . . . . . 162

Simulated time courses of product growth fOr
various values of kg, for Model 4. . . . . . . 162

Simulated saturation curve of enzyme activity
at an enzyme concentration of 0. 044‘pM, fOr
Model 4. O O O O O O O O O O O O O O O O O O O 163

Simulated time courses of product growth fOr
various otota% eggggefifioncengrations, with

M3125 , , K = O and
K'173200 ,forBMOdel 4e 0 ? e 0}]? o e e e e e o 164

Simulated time courses of product growth fOr
variogs igigisfi subsggggepfiongenggafifionsa

With 1= 5 z 9 an

K73200 w, for ”(>ng 4 e e e 05: e e e 164

Simulated time cOurses of product growth fOr

various total enzyme concentrations, with
K85000,uM, K310, ,0004pM, K5=2003pM and

K7'4OOJJM’ forMOdel .0000. see ee 0 165

Simulated time courses of product growth fOr
various initial substrate concentrations, with

K sooo,nM 3-10, 000 K =2oo and
K-17:400pM,,forModel (IMO, e ? e 0):". e e e e e 165

Simulated saturation curves of enzyme activity

at an enzvme concentration of O. O44,pM, fOr

Model 4. Fer curve A, using the same parameter
values of Figure 6-4-11, fOr curve B, using

the same parameter values of Figure 6-4-12 . . 166

Two subunit compound hysteretic enzyme model.
‘Squares are inactive states and the circles

are active states of the enzyme. . . . . . . . 168
Two subunit compound hysteretic enzyme model . 169

Simulated time courses of product growth, the
growth-and-decay of each of the free enzymes

XX

Figure

6-5-4

6-5-5.

6-5-6

6-5-7

6- 5-8

6- 5-9

6-5-10

6-5-11

6- 6-1

Page

and the enzyme-substrate complexes, for
Made]. 5. O O O O O O O O O O O O O O O O O O 0

Simulated time courses of product growth for
various total enzyme concentrations, for
"Odel S. O O O O O O O O O O O O O O O O O O .

Simulated time courses of product growth
for various initial substrate concentrations
for MOdEl 50 O O O O O O O O O 0 O O O O O 0 0

Simulated time courses of product growth for
various values of K1, for Model 5. . . . . . .

Simulated time courses of product growth for
various values of kg, for Model 5. . . . . . '.

Simulated time courses of product growth for
various values of 1:10, for Model 5 . . . . . .

Simulated time courses of product growth for
various values of 1:11, for Model 5 . . . . . .

Simulated time courses of product growth for
garious values of 1‘10, including k10=0, for
Odel 5. O O O O O O O O O O O O O O O O O O 0

Simulated saturation curve of enzyme activity
raftdag gnzyme concentration of 0.044 A", for
o e O O O O O O O O O O O O O O O O O O 0 0

Simulated saturation curves of enzyme activity
for Model 1 through Model 5 at (E)o=o,o44 )lM .

Components in the luciferase catalyzed reactio
reactions. . . . . . . . . . . . . . . . . . .

Overall spectral changes for the firefly
luciferase catalyzed reaction in 0.02514
glycylglycine pH 7.8 buffer solution at
2530.100. The initial concentrations are:
16.1 uM luciferin with 1 mM ATP and 2.5 mM

" Mg“, and 4):M luciferase. . . . . . . . . . .

Time cuts showing the decay of substrate(LH2),
the growth of enzyme-bound product and the
growth-decay of the transient intermediate for
the firefly luciferase catalyzed reactions in
$.025Moglycylglycine pH 7.8 buffer solution at
5:001 00. O O O O O O O O O O O O O O O O O O

171

173

173

174

174

175

175

176

177

179

182

186

187

Figure
7-4

7-5

7-10

7-11

8-1

8-2

8-3

Page

Time course fOr the transient intermediate(s)

in the early 23 sec for the luciferase

catalyzed reaction in 0.025M glycylglycine

pH 7.8 buffer solution at 25.010.100 . . . . . 188

Semilog plot of (A(t)-A(tm))/(A(to)-A(tw))
vs time fOr the luciferase catalyzed reaction.
The data obtained from Figure 7-3. . . . . . . 190

Semilog plot of (A(t¢)-A(t))/(A(tm)-A(to)) 173

time fOr the luciferase catalyzed reaction.
The data obtained from Figure 7-3. . . . . . . 191

Postulated sequence of events fOr luciferase
catalyzed reactions. . . . . . . . . . . . . . 189

Computer fits for 1H2 decay at various wav
wavelengths in the luciferase catalyzed

reaction, with the assumption of a

consecutive first order mechanism. . . . . . . 194

Computer fits fOr enzyme-bound product growth

at various wavelengths in the luciferase

catalyzed reaction, with the assumption of a
consecutive first order mechanism. . . . . . . 195

Computer fits fOr’IHZ decay at various

wavelengths in the luciferase catalyzed

reaction, with the assumption of a parallel

first order mechanism. . . . . . . . . . . . . 198

Computer fits fOr the enzyme-bound product

growth at various wavelengths in the luciferase
catalyzed reaction, with the assumption of a
parallel first order mechanism . . . . . . . . 199

The overall kinetic fluorescence spectra fOr

the backward reaction in 0.05M phosphate pH 7.4
buffer solution at 22.0:0.1°C. Solvent

background has been subtracted . . . . . . . . 206

.. Characteristic spectra isolated from

Figure 8-1, showing the red shift of the
fluorescence maximum of bound NADH during
the reaction . . . . . . . . . . . . . . . . . 207

Time cuts of Figure 8-1, showing the decay of

NADH during the first 3.6 sec of reaction, at
various wavelengths. . . . . . . . . . . . . . 208

xxii

Figure

8-4

8-5

9-1_

9-2

9-3

9-4

9-5

9-6a

9-6b

Page

The isolated kinetic fluorescence spectra for

the fOrward reaction in 0.05M phosphate pH 7.4
buffer solution at 22.0:0.1°C. Solvent

background has not been subtracted . . . . . . 209

Time cuts of Figure 8-4, Showing the growth
of NADH, at various wavelengths. . . . . . . . 210

Overall spectral changes fOr the ANS-

BSA binding reaction in 0.1M phosphate

pH 7.0 buffer solution at 22°C. The

initial concentrations are: 15‘pM ANS

and 3.8‘pM BSA. The spectra were Obtained

with a scanning rate of 13.33 msec. per
speCtrumeeeeeeeeeeeeeeeeeee215

Overall spectral changes for the ANS-BSA
binding reaction in 0.1M phosphate pH 7.0
buffer solution at 22.0 i 0.1 C. . . . . . . . 216

Overall fluorescence spectral changes
for two different BSA concentrations in
0.1M phosphate pH 7.0 buffer solution

at22001001oceeeeeeeeeeeeeeee217

Time development of ANS fluorescence

enhancement for the ANS-BSA binding

reaction with initial concentrations:

15).:M ANS and 3.8)iM BSA, in 0.1M

phosphate pH 7.0 buffer solution at 22°C.

The spectra were obtained from a fixed

wavelength experiment at 524 nm fOr

three successive pushes. . . . . . . . . . . . 218

Time courses showing two successive

fixed wavelength pushes at 359 nm for

the ANS-BSA binding reaction in 0.1M

phosphate pH 7.0 buffer solution at

22°C, with initial concentrations:

5 )1M ANS Md 308M. 0 O O O O O O O O O O C O 219

Initial and final fluorescence intensities
obtained at (ANS) a 15,pM, for the ANS-

BSA binding reactgon in 0.1M phosphate

PH 7.0 buffer solution at 22°C . . . . . . . . 223

Plot of'the observed first order rate

constants(k b ) vs the ratio of the
uivalent Initial concentrations,

NS) /5(BSA)O, for the binding

reaction of ANS to BSA in 0.1M phosphate

xxiii

Figure ‘ Page

pH 7.0 buffer solution at 22.0 i 0.100. . . . 224

9-7a Com uter fits fOr the binding reaction of ANS
to SA in 0.1M phos hate pH 7.0 buffer solution,
at 22.010.100, for BSA)0=3.83pM. The
concentration of ANS is SlpM. . . . . . . . . 225

9-7b Computer fits fOr the binding reactions of ANS
, to BSA in 0.1M phosphate pH 7.0 buffer solution
solution at 22.010.100, fOr (BSA)O=5‘uM and
10 . The initial concentration of NS is

5 O O O O O O O O O O O O O O O O

226

9-8a Computer fit fOr the binding reaction of ANS
to BSA in 0.1M phos hate pH 7.0 buffer solution
at 22.030.100, fOr BSA) =3.8/uM. The initial
concentration of ANS is 35 PM . . . . . . . . 227

9-8b Computer fits fOr the binding reaction of ANS
to BSA in 0.1M phos hate pH 7.0 buffer solution
at 22.030.100, fOr BSA)0=5,pM and 10 pM. The
initial concentration of ANS is 15‘pM . . . . 228

10-1 Overall kinetic absorbance spectra fOr the
redox reaction of chromic acid and hydrogen
peroxide. The reaction was carr$ed out in
0.1M ionic strength at 22.010.1 C, and was
observed with a path length of 2 mm. The
spectra were col ected with a scanning rate of
of 13.33 msec/spectrum. . . . . . . . . . . . 231

10-2 Time cuts of Figure 10-1, Showing the
disappearance of chromic acid and the growth
of peroxychromic acid(fast growth fOllowed by
a subsequent slow decomposition). . . . . . . 232

10-3 Time cuts fOr the fbrmation of peroxychromic
acid, showing the slow decomposition process
observed at 576 nm at 0.1M'ionic strength and
220030.100. 0 O O O I O O O O O O O O O O O O 233

10-4 Time cuts at various wavelengths, obtained
" from a push of the reaction of chromic acid
with hydrogen peroxide in 0.1M ionic strength
at 22.0:0.1°C. The reaction was observed
with a path length of 1.9 cm. . . . . . . . . 235

10-5 Plots of ln(A(t)-A(ta9) vs time at various

wavelengths fOr the fOrmation of peroxychromic
acid in 0.1M ionic strength at 22.010.100 . . 236

xxiv

Figure
10-6a

10-6b
10-6c
11-1
1142
11-3

11-4

11-5

11-6

11-7

11-8

Computer fits for the redox reaction of
chromic acid and hydrogen peroxide in 0.1M
ionic strength at 22.010.1 C, at wavelengths
337 nm and 352 nm. The initial concentrations
are:(HCr04)o=O.5 mM, (H202)o=7 mM and
(H+)o=14 mm o o o o o o o o o o o o o o o o 0

Computer fits at wavelengths 372 nm and
348 nm fOr the same push of Figure 10-6a. . .

Computer fits at wavelengths 544 nm and 576 nm
fbr the same push of Figure 10-6a . . . . . .

Overall kinetic absorbance spectra fbr the
AMP deaminase catalyzed reaction in 0.05M
Tris-Mes pH 6.4 buffer solution at 22.030.100

Some isolated intermediate spectra of
Figure 11-1, collected at specific time of
reaCtj-on. O O O O O O O O O O O O O O O O O 0

Time cuts at various wavelengths of
Figure 11-1, fbr the first 30 sec of the
reaCtiono O O O C O O O O O O O O O O O O O 0

Time course of the absorbance change at 287 nm
from Figure 11-1, fbr the first 14 sec of the
reaCtiono o o o o o o o 030 o o o o o o e o 0

Full time course of the AMP deaminase
catalyzed reaction, showing the growth of

IMP at 287 nm, in 0.05M Tris-Mes pH 6.4 buffer
solution at 22.010.100. . . . . . . . . . . .

Time course of'the AMP deaminase catalyzed
reaction, showing the fraction of reaction
obtained at various wavelengths from the same
ush, in 0.05M Tris-Mes, 0.2M TMACIn pH 6.4
uffer solution at 22.010.100 . . . . . . . .

Time course of the AMP deaminase catalyzed
reaction, showing the fraction of reaction
obtained at 266 nm from two successive pushes
in 0.05M Tris-Mes, 0.2M TMACL, pH 6.4

buffer solution at 22.0:0.1°C . . . . . . . .

Full time course of the AMP deaminase catalyze
catalyzed reactions, showing the dependence
of reaction progress on the initial substrate
concentration, in 0.05M Tris-Mes pH 6.4 buffer
solution at 22.010.100. The total enzyme

XXV

Page

240

241

242

247

248

249

251

252

253

254

Figure

11-9

11-10

11-11

Page
concentration is 0.209 mg/ml . . . . . . . . . 255

Full time course of the AMP deaminase catalyzed
reactions, showing the dependence of the

reaction progress on the total enzyme
concentration, in 0.05M Tris-Mes pH 6.4 buffer
solution at 22.010.100. The initial substrate
concentration is 0.025 mm. . . . . . . . . . . 256

Comparison of the computer simulated full time
course of the reaction with the experimental
data in Figure 11-6. . . . . . . . . . . . . . 258

Comparison of the computer simulated full time

course of the reaction with the experimental
data in Figure 11-7. . . . . . . . . . . . . . 259

xxvi