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This is to certify that the

thesis entitled

KINETICS OF DESORPTION OF BOVINE SERUM ALBUMIN AT THE OIL-WATER

INTERFACE USING TOTAL INTERNAL REFLECTION FLUORESCENCE AND
FLUORESCENCE PHOTOBLEACHING RECOVERY

presented by

Brian Shukla

has been accepted towards fulfillment
of the requirements for

M.S. Chemical Engineering

 

 

degree in

KEMWIW'

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Major ofessor I

 

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1198 cJCIFIC/DfiDuopGS—p.“

KINETICS OF DESORPTION 0F BOVINE SERUM ALBUMIN AT THE OIL-
WATER INTERFACE USING TOTAL INTERNAL REFLECTION FLUORESCENCE
AND FLUORESCENCE PHOTOBLEACHING RECOVERY
By

Brian Shulda

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Chemical Engineering

1998

ABSTRACT
KINETICS 0F DESORPTION OF BOVINE SERUM ALBUMIN AT THE OIL-
WATER INTERFACE USING TOTAL INTERNAL REFLECTION FLUORESCENCE
AND FLUORESCENCE PHOTOBLEACHING RECOVERY
By

Brian Shukla

The primary objective of this thesis was to measure the kinetics of desorption of bovine
serum albumin (BSA) labeled with fluorescein-S-isothiocyanate (F ITC) at the oil-water
interface. We used a S-W argon ion laser, an inverted microscope, and a photomultiplier
tube connected to a data acquisition system. We measured the dynamics of protein
desorption by total internal reflection fluorescence (TIRF) microscopy, along with

fluorescence photobleaching recovery (FPR).

Fluorescence recovery data were fit to a biexponential kinetic model to extract the rate
constants. At a bulk BSA-FITC concentration of 0.07 mM (millimoles per liter), the
apparent fast desorption rate constant (k1) was 0.52 i 0.12 s", the apparent slow
desorption rate constant (k2) had a value of 0.008 i 0.003 s", and the immobile fraction
(r0) was calculated as 0.46 i 0.04. Slow photobleaching effects were accounted for by
introducing a slow photobleaching rate constant (k p2,) into the analytical model. We
demonstrated that failure to account for slow photobleaching effects leads to an

overestimation of the immobile fraction. A low signal to noise ratio contributed to some

uncertainty in the desorption rate constants.

Dedicated to my family for their many years of love and support.

iii

ACKNOWLEDGMENTS

I would like to thank my advisor, Dr. Robert Y. Ofoli, for guiding and encouraging me
during the course of my studies and research. Thanks also to Ari Gajraj, Jamaica Prince,
and Sumei Wei for providing helpful input on my project, with additional thanks to Ari for
working diligently with me to get the equipment ready for experimentation. I would also
like to thank Parijat Jauhari, whose thesis work showed that TIRF could be used to study
protein interactions at the oil-water interface. Finally, I would like to thank the
Department of Chemical Engineering at Michigan State University for giving me this

opportunity to further my intellectual growth.

iv

TABLE OF CONTENTS

LIST OF FIGURES ........................................................................................................ Vi
LIST OF TABLES ....................................................................................................... viii
1. INTRODUCTION ................................................................................................... 1
1.1 Motivation for this study ...................................................................................... l
1.2 Objectives ............................................................................................................ 2

2. THEORY ................................................................................................................ 4
2.1 TIR excitation at a single interface ........................................................................ 4
2.2 Fluorescence Photobleaching Recovery (FPR) ...................................................... 6
2.3 Adsorption/desorption kinetics at an interface ...................................................... 6

3. EXPERIMENTAL MATERIALS AND TECHNIQUES ....................................... 11
3.1 Materials preparation ......................................................................................... 11
3.1.1 Technique for protein labeling .................................................................... 11
3.1.2 PEO coating of bottom slide ....................................................................... 12

3 .2 Experimental equipment ..................................................................................... 12
3.2.1 TIRF/FPR setup ......................................................................................... 12
3.2.2 The experimental cell .................................................................................. 17

3.3 Experimental techniques ..................................................................................... 19
3.3.1 The oil-water interface ................................................................................ 19
3.3.2 Focusing of microscope objective at the liquid-liquid interface .................... 20
3.3.3 Fluorescence photobleaching recovery (FPR) ............................................. 23

4. ANALYSIS TECHNIQUE .................................................................................... 25
4.1 A biexponential trend in the kinetics of desorption .............................................. 25
4.2 Accounting for slow photobleaching due to the monitoring beam ....................... 26
4.3 Analysis of TIRF/FPR data ................................................................................. 27

5. RESULTS AND DISCUSSION ............................................................................ 29
5.1 Desorption kinetics of BSA-FITC at an oil-water interface ................................. 29
5.2 Discussion .......................................................................................................... 49
5.3 Possible sources of error .................................................................................... 51

6. SUMMARY AND CONCLUSIONS ..................................................................... 54
7. SUGGESTIONS FOR FUTURE WORK ............................................................... 57
7.1 Protein adsorption onto a clean interface ............................................................ 57
7.2 Effect of labeling ratio on protein desorption kinetics ......................................... 6O

8. BIBLIOGRAPHY .................................................................................................. 61

LIST OF FIGURES

Figure 2.1: Total internal reflection, with resulting evanescent wave, at an oil-water
interface. The evanescent wave decays exponentially into the rarer medium. ............ 5

Figure 3.1: TIRF/FPR equipment. The laser beam is first split into photobleaching and
monitoring beams and then recombined by optical flats before being directed to the
oil-water interface. The PMT is used to collect the fluorescence emission. ............. 16

Figure 3.2: The experimental cell, or "flowcell." Note that the top view shows an "open"
cell, meaning that the prism and top slide have been removed to give an internal view
of the cell. .............................................................................................................. 18

Figure 3 .3: TIRF/FPR experiment showing one effect of improper focusing of the
microscope objective. A photobleaching pulse occurs at time 15 seconds, but no
post-bleaching drop in intensity is observed, because the microscope objective is
focused in the bulk protein solution, rather than at the oil-water interface. .............. 22

Figure 5.1: Typical fluorescence recovery curve for TIRF/FPR experiments. Initially,
there is a fast recovery of fluorescence (1), which is followed by a slower period of
recovery (2). The fluorescence does not completely recover to its pre—bleach level
(3), indicating that a fraction of photobleached proteins are irreversibly adsorbed to
the interface. F (-) is the equilibrium fluorescence prior to photobleaching, and F(O)
is the fluorescence immediately after the photobleaching flash, which occurs
approximately 10 seconds into the experiment and has a duration of 100 ms. The
bulk protein concentration for this data was 0.04 mM ............................................. 30

Figure 5.2: Drop in fluorescence during a long experiment, due to slow photobleaching by
the monitoring beam. The fluorescence level abruptly drops after the photobleaching
flash. A partial recovery of fluorescence occurs over a brief period (1), after which
the fluorescence level slowly drops, due to unintended photobleaching (2). The bulk
protein concentration for this data was 0.04 mM. ................................................... 31

Figure 5.3: Apparent rapid desorption rate constant k1 as a function of bulk protein
concentration. Each data point represents the average of twenty experiments
conducted at room temperature (~20°C) and in the dark. The photobleaching pulse
duration was 100 ms. ............................................................................................. 36

Figure 5.4: Apparent slow desorption rate constant k; as a firnction of bulk protein
concentration. Each data point represents the average of twenty experiments
conducted at room temperature (~20°C) and in the dark. The photobleaching pulse
duration was 100 ms. ............................................................................................. 38

Figure 5.5: Slow photobleaching rate constant k Pb as a function of bulk protein

concentration. Each data point represents the average of twenty experiments
conducted at room temperature (~20°C) and in the dark. The photobleaching pulse
duration was 100 ms. ............................................................................................. 40

Figure 5.6: Immobile fraction ro as a function of bulk protein concentration. Each data
point represents the average of twenty experiments conducted at room temperature
and in the dark. The photobleaching pulse duration was 100 ms. ........................... 42

Figure 5.7: Fraction of proteins (r1) that desorb at an apparent rate of k1, as a function of
bulk protein concentration. Each data point represents the average of twenty
experiments conducted at room temperature (~20°C) and in the dark. The
photobleaching pulse duration was 100 ms. ............................................................ 44

Figure 5.8: Fraction of proteins (r2) that are more tightly held at the interface and desorb
at an apparent rate of k; as a function of bulk protein concentration. Each data point
represents the average of twenty experiments conducted at room temperature
(~20°C) and in the dark. The photobleaching pulse duration was 100 ms. .............. 45

Figure 5.9: Short-term theoretical recovery curves generated fi'om Eqn. [19], using the
kinetic parameters obtained via curve fitting. The bulk concentration is 0.07 mM.
The curve that assumes absence of slow photobleaching was generated by setting k p,

equal to zero in Eqn. [19] ....................................................................................... 47

Figure 5.10: Long-tenn theoretical recovery curves generated from Eqn. [19], using the
kinetic parameters obtained via curve fitting. The bulk concentration is 0.07 mM.
The curve that assumes absence of slow photobleaching was generated by setting k p,

equal to zero in Eqn. [19]. ...................................................................................... 48

vii

LIST OF TABLES

Table 5.1: Summary of the apparent desorption and photobleaching rate constants for
BSA-FITC at the oil-water interface ....................................................................... 33

Table 5.2: Values obtained for the immobile fraction (r0), and the fractions of proteins that
desorb at rates k1 and k; (r, and r2, respectively). .................................................... 34

viii

1. INTRODUCTION

1.1 Motivation for this study

The ability to quantify the dynamic behavior of proteins at the liquid-liquid interface is of
considerable industrial importance. The food processing industry uses proteins as
emulsifiers to lower the interfacial tension between two immiscible liquid phases and
facilitate the dispersion of one phase in the other (Dickinson et al. 1990). The stability of
emulsions such as margarine and mayonnaise depends on the adsorption of proteins at the
oil-water interface. The pharmaceutical industry needs to understand the behavior of
drugs at biomembranes to determine their pharmacological activity. Biomembranes are
structurally complex, but immiscible oil-water interfaces may be used as a simplified model

in drug delivery experiments (Arai et al. 1996).

Total internal reflection fluorescence (TIRF) has been used extensively to quantify protein
behavior at the solid-liquid and membrane-liquid interfaces (Axelrod 1981; Burghardt and
Axelrod 1981; Thompson et al. 1981; Zimmerman et al. 1990; Hellen and Axelrod 1991;
Pearce et al. 1992; Pisarchick et al. 1992). TIRF is ideal for molecular-level studies of the
interfacial behavior of amphiphiles, because the fluorescence of molecules at and/or near
the interface are detected preferentially to that of molecules in the bulk liquid. This is due
to the nature of the evanescent wave formed by total internal reflection at the interface, as

will be discussed in detail in Chapter 2.

TIRF, along with fluorescence photobleaching recovery (FPR), was recently used
successfully in our laboratory to quantify the diffusion of proteins at and near an
immiscible oil-water interface (J auhan’ 1997). Jauhari’s work has provided a framework
upon which the present TIRF study is based, to study other aspects of protein dynamics at
the liquid-liquid interface. This study focuses on the kinetics of protein desorption at the

oil-water interface.

1.2 Objectives

The goal of this study is to measure the kinetics of desorption of bovine serum albumin
(BSA) labeled with fluorescein-S-isothiocyanate (F ITC) at the oil-water interface, using a
combination of TIRF and FPR. The first part of the work involves the adjustment of the
experimental TIRF apparatus to bring the system closer to the reaction limited regime (and
thus further fi'om the transport limited regime). It has been shown in previous studies
(Thompson et al. 1981; Pearce et al. 1992; Pisarchick et a1. 1992) that the system
approaches the reaction limited regime as the depth of observed fluorescence decreases

and as the bulk protein concentration increases.

The depth of the observed fluorescence is a firnction of several factors that are all
constants in our experimental apparatus (please see Chapter 2), and therefore the
fluorescence penetration depth is not readily adjustable. However, the system can be
brought closer to the reaction limited regime by determining the optimum BSA-FITC

concentration range for desorption studies. An effective method of determining whether

the system is reaction limited or not is to measure the fluorescence recovery as a function
of bulk protein concentration. If the kinetics show little or no dependence on
concentration, then the system is not limited by transport (diffusional) processes but rather
by the rates of adsorption/desorption at the interface (Pearce et al. 1992; Pisarchick et al.

1992)

The second part of the work was to determine the apparent fast and slow desorption rate
constants (k1 and k2, respectively, as outlined later in this thesis) for BSA-FITC complexes
at the oil-water interface. Optical perturbation, in the form of spot photobleaching, was
used to move the system away from equilibrium. This was accomplished by flashing an
intense beam of light briefly at the oil-water interface, causing an irreversible bleaching of
labeled proteins at and near the interface. The resulting exchange between bleached and
unbleached proteins at the interface is then monitored. The fluorescence recovery data as
a function of time can be used, as described in Chapter 2, to determine the kinetics of

desorption at the interface.

2. THEORY

2.1 TIR excitation at a single interface

When a beam of light passes through an optically transparent medium with a higher
refractive index (11;) and arrives at the interface between that medium and one of a lower
refractive index (n2), it will undergo total internal reflection (TIR) if its angle of incidence

9, measured normal to the interface, exceeds a critical angle 6,. given by

a, = sin-1(1) [11
n]

where n; < m.

Although TIR occurs for 6 > 9.- , some energy penetrates the interface in the form of an

evanescent wave (or electromagnetic field), as shown in Figure 2.1. The intensity of the

evanescent wave, which is a function of depth below the interface, is given by
2
1(2) = 10 exp(- 2) [2]

where I (z) is the intensity of light at depth 2 below the interface, 10 is the intensity at the

interface (2 = 0), and d, the evanescent wave penetration depth, is given by

2'0

d :
47ml Jsin 2 9 — (n2 /n1 )2

 

[3]

 

where 10 is the wavelength of the incident light in a vacuum.

For our system, d has a fixed value of 144 nm.

A detailed analysis of the evanescent wave, in which the electric and magnetic components

are treated in three spatial directions, is given by Axelrod, et al. (1992).

Reflected Beam

 

 

Incident Beam

Oil (refractive index 111)
Water (refractive index n2)

h

v

Evanescent Wave

  

Figure 2.1: Total internal reflection, with resulting evanescent wave, at an oil-water
interface. The evanescent wave decays exponentially into the rarer medium.

2.2 Fluorescence Photobleaching Recovery (FPR)

FPR is used to perturb the system and move it away from equilibrium. Fluorescence is
induced in BSA-FITC molecules at and near the interface via an evanescent wave
produced by a low intensity beam that we will refer to as the monitoring beam. A high
intensity beam, called the photobleaching beam, is briefly flashed at the interface,
photobleaching every fluorescent species within the field of the evanescent wave. When a
fluorescent molecule is photobleached, it permanently loses its ability to fluoresce, due to
a photochemical destruction of the fluorophore. Since the protein absorbs only small
amounts of energy at the laser wavelength (488 nm) used for photobleaching, the
fluorescent label is destroyed but not the protein. Once photobleaching has occurred, the
fluorescence signal measured by the photomultiplier tube (PMT) will partially recover
towards its prebleach level, due to the exchange of unbleached molecules in the bulk water

phase with photobleached molecules at and very near the interface.

In this study, fluorescence photobleaching recovery has been used to characterize the
kinetics of protein desorption at the oil-water interface. The fluorescence recovery profile

following photobleaching was used to obtain desorption rate constants for the system.

2.3 Adsorption/desorption kinetics at an interface

Consider labeled amphiphilic molecules of bulk concentration [A] in equilibrium between
an interface-bound state and a free solute state. The molecules can adsorb to free binding

sites of interface concentration [B] to form complexes of interface concentration [C], or

the molecules can desorb from the complexes back into the bulk solution. This process

can be represented as a chemical reaction of the form

k__a)

A(r, z ,z) + B(r, t) —’,C(r t) [4]

where kc and k d are the adsorption and desorption rate constants, respectively, r is the

radial distance from the interface observation area to another point on the interface, 2 is
the perpendicular distance from the interface observation area to a point in the bulk, and t

is time.

In this context, binding refers to the adsorption of a molecule from the bulk solution onto

an available site (or location) on the interface. The equilibrium of the system is described

by

c
73 . m

ae- law:-

where Z , B , and C are the equilibrium concentrations (bleached and unbleached) of A,

B, and C, respectively.

Thompson et a1. (1981) derived a mathematical model for use with TIRF/FPR

experiments. A condensed version of their derivation is given below.

The differential equations governing A(r, z, t) and C (r, t) are, respectively

-%=EWM m

and

fg—Ct-zpcvfmk A B—de [7]

a z—)0

where D A and DC are the bulk and interface diffusion coefficients, respectively, V3 and
Vi, are two and three dimensional Laplacian operators, respectively, and

A oslim

Z-)

A(r, z, t) .

Z-‘)0

The task now is to relate fluorescence intensity to the interfacial concentration of proteins.

Thompson et al. (1981) showed that, following photobleaching

F(—) — F(t) = Q1. jut/mi? — C(r,t))d2r [8]
where F (—) is the equilibrium fluorescence prior to photobleaching, F (t) is the
fluorescence at any time after the photobleaching flash (which occurs at t = 0 by
definition), Q is a product of the efficiencies of excitation light absorption and
fluorescence emission and detection and is usually referred to as the quantum yield, [0 is
the maximum evanescent intensity at the interface, and I/I(r) is a dimensionless evanescent

intensity profile function with a maximum value of one.

After integrating Eqn. [8] and incorporating the initial and boundary conditions,
Thompson et al. (1981) used linear transformation theory to obtain the following

analytical solution:

F(—) — F(t) = F53 : {fl/(2°) v1/2w(— i,/v,k,r )— v1/2w(— i,/v_k,z)] [9]

 

where

 

vi,” =1 1L(—1:,/1—4R,M,/kd) [10]
2 Ram;
w(i77) = e”2 erfc(n) (77 complex) [11]
and
Rm) 5 —_D—:— (the " bulk normal diffusion rate") [12]
(C /A)2

Equation [9] is impractical to use with experimental data, not only because of its
complexity, but because it contains both bulk diffusional and desorption effects which will
not be readily distinguishable in a TIRF/FPR experiment. However, if the desorption rate

k d is much smaller than the bulk normal diffusion rate R my , then the system will be in the

reaction limited regime, and the functional dependence of Eqn. [9] on the bulk diffusion

coefficient, D .4 , will be negligible.

It has been shown in previous studies (Thompson et al. 1981; Pearce et al. 1992;
Pisarchick et a1. 1992) that increasing the bulk protein concentration brings the system
closer to the reaction limited regime. Burghardt and Axelrod (1981) showed that, for bulk
concentrations of BSA greater than or equal to 0.015 mM (millimoles per liter),
fluorescence recovery is controlled by desorption reaction kinetics rather than by bulk
diffusion. Under such conditions, Eqn. [9] reduces to the approximate form (Thompson et

al. 1981)
F(—) — F(t) z [F(—) - none” [13]
Equation [13] represents the general form of the mathematical model used to analyze the

TIRF/FPR data in this study. However, further modifications were made to this equation

to make it more appropriate for this work. These modifications are discussed in Chapter

4.

10

3. EXPERIMENTAL MATERIALS AND TECHNIQUES

3.1 Materials preparation

3. 1. 1 Technique for protein labeling

Bovine serum albumin (BSA), essentially fatty acid-free (A-7511, molecular weight
~67,000 g/mol), was obtained from the Sigma Chemical Co. (St. Louis, MO) and labeled
with fluorescein-S-isothiocyanate (F -1907, molecular weight 389 g/mol, Molecular
Probes, Eugene, OR) to form a BSA-FITC complex. FITC was covalently bonded to the
lysine groups of BSA. All labeling reactions were done in a 0.10 M borax buffer (B-9876,
Sigma Chemical Co., St. Louis, M0) for four hours at room temperature and pH ~9.2 in

the dark, following the procedure described by Lok et al. (1983a).

To separate unconjugated FITC from BSA-FITC, the protein solution was dialyzed with
stirring against a 0.03 M phosphate-buffered saline (PBS) buffer for 24 hours at pH ~7.4,
using molecularporous regenerated cellulose dialysis membrane (Spectra/POI 1, molecular
weight cutoff = 6,000, The Spectrum Companies, Gardena, CA). The solution was then
dialyzed a second time for 24 hours using a second PBS buffer solution to further remove
unconjugated F ITC. During the dialysis process, PBS buffer exchanges with borax buffer,
so that the protein solution within the dialysis bag is mostly PBS, and the pH is close to
7.4 as desired for the experiments conducted in this study. All buffers were made with

deionized water from the NAN Opure ultrapure water system (Bamstead, Dubuque, IA).

11

The labeling ratio of the protein, which is defined as the molar ratio of conjugated FITC to
BSA, was determined spectrophotometrically using absorbance measurements at 280 nm
for BSA and 488 nm for FITC. We used labeling ratios low enough to avoid
concentration quenching, a phenomenon whose causes and effects were discussed in detail

by Robeson (1995).

3. 1.2 PE 0 coating of bottom slide

In order to prevent adsorption of protein molecules on the bottom interface of the
experimental cell, the bottom slide (i.e. the bottom of the flowcell) was coated with
poly(ethylene oxide) (PEO) (Cheng et al. 1987). Deionized water was used to make a 0.5
wt% PEO solution. A bottom glass slide, which had previously been cleaned using the
procedure described by Cheng et al. (1987), was immersed in the PEO solution for two
hours, thereby forming a PEO coating on the slide. Before the slide was used, it was

rinsed with water to remove any excess PEO.

3.2 Experimental equipment

3. 2. r TIRF/FPR setup

The experimental setup used in our lab for TIRF/FPR experiments consists of a S-W Lexel
Model 95 continuous wave argon ion laser (Lexel Lasers, Inc., Fremont, CA), an Axiovert
135M inverted microscope (Carl Zeiss, Inc., Thomwood, NY), a photomultiplier tube

(model R4632, Hamamatsu Corporation, Bridgewater, NJ) jacketed in a thermoelectric

12

cooling system (TE177TSRF, Products for Research, Inc., Danvers, MA) to increase the
signal-to-noise ratio, a CCD camera (MTI, VE1000, Carl Zeiss, Inc., Thomwood, NY), a
modular automation controller (MAC 2000, Ludl Electronic Products Ltd, Hawthorne,
NY) with a computerized data acquisition system (Viewdac, Keithley Instruments, Inc.,
Rochester, NY), and a double-syringe pump (Model 551382, Harvard Apparatus, South
Natick, MA). The entire setup sits on a vibration isolation table (RS 4000, Newport
Corporation, Irvine, CA), which darnpens vibrations from external sources, to mitigate

against excessive mobility of the oil-water interface.

The laser, which operates at a wavelength of 488 nm, is attenuated using neutral density
filters (03FNQ, Melles Griot, Boulder, CO). The original beam is first split into two
beams (a monitoring beam and a photobleaching beam) of vastly unequal intensities, using
an optical flat, and then recombined using a second optical flat. This arrangement ensures
that the monitoring and photobleaching beams are perfectly aligned after recombination
(Thomas and Webb 1990). In between the optical flats where the beam is split into two
parts, a neutral density filter is used to further attentrate the monitoring beam. In addition,
a programmable shutter (D122, Uniblitz, Vincent Associates, Rochester, NY) is placed in
the path of the photobleaching beam. A hole cut in the shutter ensures that the monitoring
beam is always incident at the oil-water interface, leaving the photobleaching beam

shuttered, except during the brief instances during which photobleaching is required.

The monitoring beam, whose power is approximately 15 uW before entering the prism,

provides a light level sufficient to excite the fluorophores at the interface without inducing

l3

unacceptable levels of photobleaching. When the shutter is opened, the recombined
photobleaching and monitoring beams strike the interface at the same location. The
photobleaching beam, whose power is approximately 100 mW before entering the prism,
provides a light level strong enough to photochemically destroy the fluorophores
(photobleaching). A photobleaching pulse duration of 100 ms was used for most

experiments.

The recombined beam is passed through a half wave polarizer (10RP02-12, Newport
Corporation, Irvine, CA), to enable a choice of either horizontal or vertical polarization of
the incident light. In addition, an optical chopper (SR540, Stanford Research Systems,
Inc., Sunnyvale, CA) is available during lengthy experiments to reduce unintended
photobleaching by the monitoring beam. The beam is directed to the interface via a series
of mirrors and focused onto the interface using a plane-convex lens (f = 200 mm, Oriel
Corporation, Stratford, CT). It strikes the interface within the experimental cell at an

angle of 64°, measured normal to the interface.

Fluorescence emission is collected by a photomultiplier tube (PMT) through a microscope
objective (440651, 32X, 0.4, Carl Zeiss, Inc., Thomwood, NY), which is focused on the
interface using the method described in section 3.3.2. A narrow band filter (535DF35,
518-552 nm wavelength range, Omega Optical, Inc., Brattleboro, VT) is placed below the
microscope objective so that only light within the wavelength range of the emitted

fluorescence can reach the PMT. The light that passes through the filter is directed

l4

through the microscope to the PMT where its intensity is measured and converted into a

digital signal.

The experimental setup used in our lab for TIRF/FPR experiments is shown in Figure 3.1.

15

 

 

F1

 

NDF "
m

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

9?
o 2.
a

3

1:: a .5

g“

o. 8 §;
23
EE
0 :O‘
E If: as
E a F .. 514‘?
° i3 cres- 2.5:
a as ea
,8 6g
if"?
——L_ a...
g ASE—ll 3 egg.
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s 87:
a an
8 (3%

U

 

 

 

Figure 3.1: TIRF/FPR equipment. The laser beam is first split into photobleaching and
monitoring beams and then recombined by optical flats before being directed to the oil-
water interface. The PMT is used to collect the fluorescence emission.

3.2.2 The experimental cell

The experimental cell used in our lab for TIRF/FPR experiments is shown in Figure 3.2.
A 64° dovetail prism is optically coupled to the top microscope slide (Fisher Scientific,
Pittsburgh, PA) using an immersion oil (16482, type A, R.P. Cargille Laboratories, Inc.,
Cedar Grove, NJ). The underside of the top slide is coated (as described later in section
3.3.1) with the same oil to form the top half of the oil-water interface. Situated below the
top slide is an aluminum spacer (1 mm thick) with a channel in the center to hold the water
or protein solution which forms the bottom half of the oil-water interface. In this study,
the terms “water” and “protein solution” are used interchangeably because the protein
solution is composed mostly of PBS buffer, which in turn is made up mostly of deionized

water.

An O-ring (Parker Seals, Lexington, KY) is placed around the perimeter of the spacer
channel to prevent leakage of the protein solution. The bottom slide is coated with PEO,
as discussed in section 3.1.2, and has two holes drilled in it to allow the inflow and
outflow of protein solution fi'om the double-syringe pump. After the cell is assembled as
shown in the side view of Figure 3.2, the assembly is held in place by screwing it onto an
anodized aluminum shell that sits on top of the microscope stage. The cell is sometimes
referred to as a “flowcell,” but it should be noted that flow is induced only during the
infusion of buffer or protein solution into the cell. All TIRF/FPR experiments in this study

were performed under stopped-flow conditions.

17

Side View of closed cell:

Pnsm
Reflected Laser Light / Incident Laser Light

 

Or'l- Water Interface

 

 

 

 

 

 

 

,Top Glass Slide
\ \/ A
Metal Spacer—b \—\/\ Protein Solution
X0410. 38 ‘ t \o r Giass Sfidc
Teflon ' J I
Inflow ”’7
Outflow

T0 SmerlP /

Top View of open cell: pm w

 

 

 

 

 

 

Protein Solution upended View ofthe til-m irrterhce
Flow Inlet Flow Outlet
L I
\ / \
__. o. :
Metal Spacer (L J 1 mg

 

 

 

 

Figure 3.2: The experimental cell, or "flowcell." Note that the top view shows an "open"
cell, meaning that the prism and top slide have been removed to give an internal view of
the cell.

18

3.3 Experimental techniques

3. 3. 1 The oil-water interface

The structure of the oil-water interface should be as reproducible as possible to ensure
that experiments are conducted under similar conditions. A different slide coating
technique than was used by Jauhari (1997) was implemented to better fulfill this important
criterion. A top slide (1 inch x 3 inches) was cleaned using the procedure described by
Cheng et al. (1987). A drop of the immersion oil described in section 3.2.2 was then
placed at one end of the slide, along its one-inch dimension. A cylindrical glass rod was
smoothly dragged over the slide, thereby sweeping the surface of the slide and evenly
coating it with oil. The oil layer was estimated to be approximately 80 um thick by
weighing multiple slides before and after coating. Since the cell has a total depth of 1 mm,
the protein solution is estimated to be 920 pm thick. Jauhari (1997) found that this depth

is thin enough to give an equilibrium adsorption time on the order of minutes.

Once the oil half of the interface has been formed on the top slide, the other side of the
slide is optically coupled to the prism using the same oil. The prism-slide assembly is then
placed, slide side down, on top of the cell, which has been pre-filled with protein solution

(the water half of the interface), to form the oil-water interface.

The protein solution is introduced into the cell using a two-step process. First, the
solution is pipetted into the open cell. The cell is then sealed by placing the prism-slide

assembly on top of it. Small air bubbles sometimes get trapped in the cell during the

19

process of sealing it. The air bubbles are subsequently removed by pumping additional
protein solution into the cell, using a double-syringe pump system which simultaneously
infuses and withdraws fluid from the cell. The exit port of the cell has been designed to

effectively aid the removal of trapped air bubbles.

We have always exercised appropriate caution when using the syringe pump, because of
the possibility of shearing oil and/or adsorbed proteins off the interface as fluid flows
through the cell. Jauhari (1997) reported that, for our experimental apparatus, a flow rate
of 0.17 mL/min can be safely used without damaging the oil-water interface or sweeping
adsorbates away. As an added precaution, we used flow rates no higher than 0.05
mL/min. Additionally, all desorption experiments were performed under stopped-flow

conditions.

3. 3.2 Focusing of microscope objective at the liquid-liquid interface

Focusing the microscope objective on the oil-water interface has been a challenge since
the beginning of our studies. There was no reproducible method of getting a proper
focus, because the oil-water interface provides no landmark on which to focus, and the oil
layer cannot be guaranteed to be the same thickness for different constructions of the oil-
water interface. Studies involving the solid-liquid interface achieve proper focus by
placing a mark, such as a small dot, on the slide and visually focusing the microscope on
that point. Jauhari (1997) previously attempted to focus using a visual technique, but the

qualitative nature of this technique could not assure a reproducible focus.

20

As a result, devising and implementing a precise method of focusing the microscope
objective at the oil-water interface was a key activity for this study. Several experiments
showed that poor focusing could lead to an inability to detect fluorescence recovery afier

photobleaching.

Zimmerman et al. (1990) observed that the exchange of proteins within the bulk occurs at
a faster rate than the time resolution of the experiment. In other words, proteins within
the bulk exchange rapidly enough that a fluorescence recovery profile will not be observed
in the experimental data; instead, the fluorescence intensity will apparently return
instantaneously to the pre-bleach level. This is apparent in Figure 3.3, which shows a
photobleaching experiment in which the microscope objective has been focused in the bulk
aqueous phase instead of at the interface. About 15 seconds into the run, a
photobleaching pulse is observed in the form of a sharp increase in the signal. When the
pulse ends, the fluorescence signal returns instantaneously to the pre-bleach level,
indicating that the fluorescence recovery occurs too rapidly for our data acquisition system

to resolve a recovery curve.

It should be noted that this type of situation can also occur when the spot being
photobleached is not properly centered in the field of view. Therefore, both proper laser
beam alignment and precise focusing of the microscope objective are essential to obtaining

useful post-photobleaching recovery profiles.

21

 

1200

 

 

 

 

 

 

 

 

 

311ooa
5
E1000“
.8;
:§ 900
S
3 800
e:
8
a 700 ~
§
E 600-
500 . , , .
o 5 1o 15 2o 25 30

Time (s)

 

 

 

Figure 3.3: TIRF/FPR experiment showing one effect of improper focusing of the
microscope objective. A photobleaching pulse occurs at time 15 seconds, but no post-
bleaching drop in intensity is observed, because the microscope objective is focused in the
bulk protein solution, rather than at the oil-water interface.

22

We have developed a new technique to obtain a more reliable and reproducible focus of
the microscope objective on the oil-water interface. We take advantage of the fact that
the intensity of the evanescent wave is highest at the oil-water interface, which is where
we want the objective to be focused. With the monitoring beam striking the interface, the
microscope objective is moved to its highest point, so that it will be focused above the
interface. The objective is then moved downward in small increments, and the light level
is checked at each increment. As the objective gets progressively lower, the light level
increases quickly and then reaches a maximum. The light level decreases as the objective
is lowered further, indicating that the oil-water interface has been passed. Moving the
microscope objective back to the initial point of the highest fluorescence signal allows us

to obtain a reliable and reproducible objective focus at the interface.

3. 3.3 Fluorescence photobleaching recovery (FPR)

FPR has been used extensively to study the kinetics of desorption at a solid-liquid
interface (Thompson et al. 1981; Burghardt and Axelrod 1981; Pearce et al. 1991;
Pisarchick and Thompson 1991; Hellen and Axelrod I991; Abney et al. 1992; Pisarchick

et al. 1992). The same technique is used in this study, essentially without modification, as

described below.

Labeled proteins at a spot on the oil-water interface fluoresce due to excitation energy
from the evanescent wave created by the monitoring beam. A brief pulse (100 ms for

most experiments) of the photobleaching beam strikes the same spot on the interface,

23

photobleaching the fluorophores at and near the interface and causing a drop in
fluorescence signal. The recovery of the fluorescence signal towards the pre-bleach level
is monitored, as photobleached molecules desorb from the interface and are replaced by

unbleached molecules fi'om the bulk solution.

In a typical TIRF/FPR experiment, 5000 data points are taken at a rate of 82 data points
per second, corresponding to an experimental run time of about one minute. Each data
point is an average of 256 readings from the photomultiplier tube. Approximately 10
seconds afier the data acquisition has begun, a 100 ms photobleaching pulse impinges on
the spot being monitored, causing the fluorophores at and near the interface to be
photobleached. The remaining 50 seconds of data acquisition records the recovery of
fluorescence due to exchange of bleached molecules at the interface with unbleached

molecules from the bulk.

A competing factor in this process is the decrease in fluorescence over time due to a Slow
photobleaching of fluorophores at and near the interface. This is a result of the continuous
impingement of the monitoring beam at the interface. If this becomes significant, then it
must also be taken into consideration in data analyses, as explained in Chapter 4, section

4.2.

24

4. ANALYSIS TECHNIQUE

4.1 A biexponential trend in the kinetics of desorption

Under reaction limited conditions, fluorescence recovery afier photobleaching is
controlled by the rate of desorption of photobleached proteins from the interface, and it
can be described by the following monoexponential equation (Thompson et al. 1981):

F(—)—F(t)~ [F(—)—F(0>]e"‘:‘ [14]
However, studies using both BSA (Burghardt and Axelrod 1981) and other proteins
(Pearce et al. 1992; Pisarchick et al. 1992) have shown that recovery curves fi'om
TIRF/FPR experiments in the reaction limited regime are best described by two
exponential desorption terms. Equation [14] can thus be rewritten in biexponential form
as (Burghardt and Axelrod 1981):

F(—) - F(t) = [F(—) - F(O)](r0 + rle'k" + r,e"‘2') [15]

where
ro+r1+r2=1 [16]

and k1 and k2 represent the apparent rapid and slow rates of desorption, respectively

(Burghardt and Axelrod 1981).

The parameters r1 and r2 represent the fraction of proteins that desorb according to rate
constants k1 and k2, respectively. The fraction of proteins that are irreversibly adsorbed to
the interface (the immobile fraction) is given by re. Therefore, the mobile fraction, f, is

given by

25

f=1—r(,=r,+r2 [17]

4.2 Accounting for slow photobleaching due to the monitoring beam

In an ideal situation in which photobleaching due to the monitoring beam does not occur,
Eqn. [15] is adequate to model fluorescence recovery in TIRF/FPR experiments. In
practice, however, a decay in signal level occurs, due to the continuous excitation of
fluorophores by the monitoring beam. This decay can be described by an exponential
function (Hirschfeld 1976; Wells et a1. 1989, Song et al. 1995) of the form

F(t) = F(0)e"‘~‘ [18]

where kph is the rate constant describing photobleaching due to continuous fluorophore

excitation by the monitoring beam.

We conducted experiments in which the monitoring beam was allowed to continuously

excite fluorophores at and near the oil-water interface and measured an average k p, value

of approximately 8x104 s". At this rate, the fluorescence level drops by about 5% over a
period of 60 seconds. Since a drop in the fluorescence signal will counteract the effects of
fluorescence recovery after spot photobleaching (FPR), we had to modify Eqn. [15] to
ensure that the effects of both desorption and photobleaching are accounted for. The

modified equation is

F(—) — F(t) = [F(—) — F(0)e"""](r0 + rle‘k" + rze‘”") [19]

26

For this equation to be valid, it must reduce to Eqn. [15] in the limit as k p, approaches
zero; it must also reduce to Eqn. [18] in the limit as both k, and k2 approach zero. It can

be readily verified that these limits are met by Eqn. [19]. Further verification of Eqn. [19]

can be based on comparing the value of k p, obtained in experiments for which no
photobleaching pulse is used (i.e. curve fitting data to Eqn. [18]) to the value of k pb

obtained in TIRF/FPR experiments (i.e. curve fitting data to Eqn. [19]). As will be
discussed further in Chapter 5, the average k p, value obtained by using both methods was

approximately 8x10’4 5", further confirming the validity and appropriateness of using Eqn.

[19].

4.3 Analysis of TIRF/FPR data

Fluorescence data are collected as described in section 3.3.3. The first 10 seconds of the
data (prior to impingement of the photobleaching beam) give the pre-bleach fluorescence

intensity, F (—). Time t = 0 is defined as the time at which the photobleaching pulse is

terminated and fluorescence recovery begins. F (0) is the fluorescence intensity at time t =
0 and can be obtained directly from the experimental data or can be determined in the

curve fitting procedure.
Using the graphing program SigmaPlot (Jandel Corporation, San Rafael, CA), the

recovery data starting at t = 0 is fit to Eqn. [19], using a nonlinear regression procedure.

The routine takes the experimental data of F (t) versus 1 and obtains a best fit curve by

27

iterating from initial guesses of F (0), r0, r1, r2, k1, kg, and k Pb values. The iterative process

is subject to two constraints: a) Eqn. [16] must be satisfied, and b) all the estimated
parameters must have non-negative values, because (as Eqn. [19] is written), negative

parameters have no physical meaning.

28

5. RESULTS AND DISCUSSION

5.1 Desorption kinetics of BSA-FITC at an oil-water interface

Four important features (or periods) can be extracted from the fluorescence recovery
curves for the desorption of BSA-FITC molecules fi'om the oil-water interface. The first
is a period of fast initial recovery, which is indicative of a fiaction (r1) of molecules that
desorb rapidly from the interface at an apparent rate la. The second feature is a period of
slower recovery, which indicates a fraction (r2) of molecules that desorb at a slower rate
(kg). The third feature is a fraction (r0) of molecules that are irreversibly adsorbed at the
interface. It is important to note that “irreversibility” in this sense only applies over the
observation time of the experiment, and that a nearly complete fluorescence recovery may
be seen if the interface is monitored over several hours. Figure 5.1 shows these first three

features.

The final important feature observed is a decay in fluorescence intensity due to the slow
photobleaching of fluorophores at the interface, induced by the continuous impingement of

the monitoring beam on the interface. This occurs at a rate k pb , and its influence on the

system becomes more pronounced at longer durations (on the order of 103 minutes).

Figure 5.2 illustrates this final feature.

29

 

 

 

 

 

Fluorescence (Arbitrary Units)

 

 

 

4100 4——— Photobleachrng flash
4000 if . . . .
0 10 20 30 40 50 60
Time (s)

 

 

 

Figure 5.1: Typical fluorescence recovery curve for TIRF/FPR experiments. Initially,
there is a fast recovery of fluorescence (1), which is followed by a slower period of
recovery (2). The fluorescence does not completely recover to its pre-bleach level (3 ),
indicating that a fi'action of photobleached proteins are irreversibly adsorbed to the
interface. F (-) is the equilibrium fluorescence prior to photobleaching, and F (0) is the
fluorescence immediately after the photobleaching flash, which occurs approximately 10
seconds into the experiment and has a duration of 100 ms. The bulk protein concentration
for this data was 0.04 mM.

30

 

300

 

N
a)
O

260

 

240 ~

220 ~

 

Fluorescence (Arbitrary Units)

N
O
O

Photobleaching flash

 

 

180

 

0 1000 2000 3000 4000 5000
Time (s)

 

 

 

Figure 5.2: Drop in fluorescence during a long experiment, due to slow photobleaching by
the monitoring beam. The fluorescence level abruptly drops after the photobleaching
flash. A partial recovery of fluorescence occurs over a brief period (1), after which the
fluorescence level slowly drops, due to unintended photobleaching (2). The bulk protein
concentration for this data was 0.04 mM.

31

Table 5.1 and Table 5.2 show the kinetic desorption parameters calculated from the
TIRF/FPR data. These variables were obtained by curve fitting the experimental data to
Eqn. [19]. Figure 5.3 through Figure 5.8 Show the results graphically as a function of bulk
concentration. The error bars correspond to the standard deviations of the average values
obtained for each parameter. Four BSA-FITC bulk concentrations were studied, ranging

from 0.01 to 0.10 mM.

Burghardt and Axelrod (1981) showed that for bulk concentrations of BSA greater than
or equal to 0.015 mM, fluorescence recovery is limited by desorption reaction kinetics
rather than by bulk diffusion. For the system to be in the reaction limited regime, they
showed that the following relationship must be satisfied:

BE”?- 2 20 [20]

d

where RBND is given by Eqn. [12].

To calculate RBND, the concentration of proteins at the interface [C] must be known.

Unfortunately, at the present time, we have no reliable technique for using fluorescence
emission data to calculate the concentration of proteins at the liquid-liquid interface.
Therefore, we have assumed that the ratios of interfacial to bulk protein concentrations at
the liquid-liquid interface are comparable to those at the solid-liquid interface. Based on
this assumption, we can use the criterion established by Burghardt and Axelrod (1981).
Thus, we expect the desorption rates to stabilize as the bulk concentration goes above

0.015 mM, and we look for diffusional effects to become negligible.

32

Table 5.1: Summary of the apparent desorption and photobleaching rate constants for
BSA-FITC at the oil-water interface.

 

 

 

 

 

 

Bulk Conc. (mM) k. (s") k, (s'l) kp. (s")
0.01 0.20 :I: 0.02 0.0015 i 0.0004 0.0011 i 0.0002
0.04 0.39 i 0.15 0.008 i 0.002 0.0009 1 0.0003
0.07 0.52 i 0.12 0.008 i 0.003 0.0007 :1: 0.0002
0.10 0.51 i 0.09 0.009 i 0.002 0.0006 1- 0.0003

 

 

 

 

 

33

Table 5.2: Values obtained for the immobile fraction (r0), and the fractions of proteins that
desorb at rates k1 and k2 (r1 and r2, respectively).

 

 

 

 

 

Bulk Cone. (mM) ro n r;
0.01 0.45 i 0.07 0.098 i 0.039 0.45 i 0.03
0.04 0.51 i: 0.15 0.093 i 0.008 0.40 i: 0.16
0.07 0.46 i 0.04 0.091 i 0.009 0.45 i 0.05
0.10 0.38 i 0.07 0.24 i 0.02 0.38 i 0.05

 

 

 

 

 

 

34

Figure 5.3 illustrates the dependence of the apparent rapid desorption rate constant k1
(units of s") on the bulk concentration. The biexponential desorption kinetic model we
have used gives a consistently good fit to the experimental data, as it has been shown to
do in studies of similar protein systems (Burghardt and Axelrod 1981; Pearce et al. 1992;
Pisarchick et al. 1992). Given the level of noise and variability in the fluorescence
recovery curves, adding a third desorption rate constant would likely not yield any more

reliable or useful information about the adsorption and desorption processes.

In this study, we define k1 as the apparent rate at which loosely adsorbed proteins desorb
from the interface. Since the proteins are loosely bound to the interface, they can more
readily desorb, and (as will be shown shortly) the k1 values were about two orders of
magnitude larger than those calculated for k2, which we classify as the apparent rate at

which proteins adsorbed directly to the oil-water interface desorb from the interface.

Figure 5.3 shows that, as the bulk concentration goes above 0.01 mM and the reaction
limited regime is reached, k1 increases and appears to quickly stabilize. At a concentration
of 0.01 mM (the first data point), the rate of fluorescence recovery is limited by bulk
diffusive processes. As the bulk concentration is increased, bulk proteins near the
interface are more readily available to exchange with proteins adsorbed at the interface,
and the exchange becomes limited by the rate at which proteins can desorb from the

interface and create room for arriving molecules.

35

0.9

 

0.8 ~

0.7 J

0.6 -

0.5 4

k, (3")

0.4 -

0.3 -

 

 

0.2 -

0.1 -

 

 

0.0 I I I I
0.00 0.02 0.04 0.06 0.08 0.10 0.12

 

Bulk Concentration (mM)

Figure 5.3: Apparent rapid desorption rate constant k, as a fimction of bulk protein
concentration. Each data point represents the average of twenty experiments conducted at

room temperature (~20°C) and in the dark. The photobleaching pulse duration was 100
ms.

36

Figure 5.4 illustrates the dependence of the apparent slow desorption rate constant k;
(units of s") on the bulk concentration. We define k; as the apparent rate at which
proteins that are more tightly adsorbed to the oil-water interface desorb from the interface.
As would be expected, these proteins desorb much more slowly than the proteins
described by k1. For that reason, the k; values are about two orders of magnitude smaller

than those of It}.

In spite of their quantitative differences, we see the same general trend in Figure 5.4 that
was observed in Figure 5.3: as the bulk concentration goes above 0.01 mM and the
reaction limited regime is reached, It; increases dramatically as it approaches an apparent
but not well-established plateau. The value of k2 increased fivefold as the bulk
concentration increased from 0.01 mM to 0.04 mM. We interpret this as an indication
that a transition between the diffirsion and reaction limited regimes has most likely

occurred, because the apparent rate of desorption is less limited by bulk protein diffusion.

37

0.016

 

0.014 —

0.012 -

0.010 -

0.008 -

k2 (s")

 

 

0.006 -

 

0.004 -

0.002 _

 

 

 

 

0.00 0.02 0.04 0.06 0.08 0.10 0.12
Bulk Concentration (mM)

Figure 5.4: Apparent slow desorption rate constant k; as a function of bulk protein
concentration. Each data point represents the average of twenty experiments conducted at

room temperature (~20°C) and in the dark. The photobleaching pulse duration was 100
ms.

38

Figure 5.5 shows the values obtained for k pb (units of s"), which measures the rate of

slow unintended photobleaching caused by the monitoring beam. It should be noted that,

for a given bulk concentration, the absolute value of k p, depends on the monitoring beam

intensity during the experiment. We conducted all experiments with a monitoring beam
intensity of approximately 15 11W (measured just before the beam enters the prism). More

interesting than the absolute value of k pb is the monotonic decrease in its value with

increasing bulk concentration. This trend is to be expected because, as the bulk
concentration increases, more fluorophores are available within the field of the evanescent
wave at any given time. Therefore the average excitation energy imparted to an individual
fluorophore decreases, accompanied by a corresponding decrease in the overall rate of

photobleaching.

For the monitoring beam intensity used in our experiments, k p, is on average about one

order of magnitude smaller than the apparent slow desorption rate constant k2. Therefore,
at first glance, it might seem unnecessary to include the effects of photobleaching in our
data analysis. However, it will be shown later that the effects of slow photobleaching are

significant, especially over longer experiments (on the order of 103 seconds).

39

 

1.6
1.4-
1.24

1.0a

 

0.8 -*

 

kp, x 103 (5")

0.6 -

 

 

0.4 “ A

0.2 a

 

 

 

0.0 i I I I
0.00 0.02 0.04 0.06 0.08 0.10 0.12

Bulk Concentration (mM)

Figure 5.5: Slow photobleaching rate constant k W as a function of bulk protein

concentration. Each data point represents the average of twenty experiments conducted at
room temperature (~20°C) and in the dark. The photobleaching pulse duration was 100
ms.

40

Figure 5.6 shows the dependence of the immobile fraction r0 on the bulk concentration.
Although the trend is not monotonic, r0 seems to decrease (within experimental
uncertainty) with increasing bulk concentration. This trend was also observed by
Burghardt and Axelrod (1981) for BSA adsorbed to a glass-water interface. Burghardt
and Axelrod (1981), however, observed r0 values that were consistently higher for a given
bulk BSA concentration than those obtained in this study at the oil-water interface. For
example, at a bulk concentration of 0.10 mM, they obtained an r0 value of about 0.5
(Burghardt and Axelrod 1981), compared to the value of 0.38 i 0.07 obtained in this

study.

This would appear to indicate that BSA molecules adsorb less irreversibly at an oil-water
interface than at a glass-water interface, which is a reasonable explanation for the
observation. One might claim that this is because an oil-water interface is less stable than
a glass-water interface and its interfacial integrity is therefore more easily compromised.
However, we have attempted to minimize vibrational effects by placing our experimental
apparatus on an excellent vibration isolation table. In addition, we have designed our
system to accommodate a thin oil layer (~80 11m) and a thin aqueous layer (~920 um),
both dimensions selected in our efforts to make the oil-water interface behave
macroscopically like a solid-liquid interface. A more plausible explanation for the
difference is that not accounting for the slow photobleaching due to the monitoring beam
can lead to an overestimation of the immobile fraction This will be discussed more fully

later in this thesis.

41

1.0

 

0.9 l
0.8 4
0.7 «

0.6 -

 

0.4 -

0.3 ~

0.2 ~

0.1 ‘

 

 

0.0 1 f u r
0.00 0.02 0.04 0.06 0.08 0.10 0.12

 

Bulk concentration (mM)

Figure 5.6: Immobile fraction r0 as a function of bulk protein concentration. Each data
point represents the average of twenty experiments conducted at room temperature and in
the dark. The photobleaching pulse duration was 100 ms.

42

Figure 5.7 shows the dependence of r1, the fraction of proteins that desorb at rate k1, on
the bulk concentration. The value of r1 stays constant at about 0.09 for bulk
concentrations between 0.01 mM and 0.07 mM, but an abrupt increase occurs between
0.07 mM and 0.10 mM. Burghardt and Axelrod (1981) did not observe this abrupt
increase at 0.10 mM BSA-FITC at the solid-liquid interface but rather saw a steady
increase in the value of r1 with increasing bulk concentration. We currently do not have a
reasonable explanation for the increase, but it is likely not due to differences between the

interface used in this study and that used by Burghardt and Axelrod (1981).

Figure 5.8 shows the dependence of r2, the fraction of proteins that desorb at rate k;, on
the bulk protein concentration. Within experimental uncertainty, r; is relatively constant
with respect to bulk concentration. Burghardt and Axelrod (1981) observed an increase in
r; with increasing bulk concentration. Such a trend might have been observed in this study
had we not encountered the abrupt increase in r1 between bulk concentrations of 0.07 mM
and 0.10 mM. The absence of this abrupt increase in r1 would increase r; at 0.10 mM

above its current value of 0.38 i 0.05.

43

 

0.30

0.25 -

0.20 d

C- 015 ‘

 

0.10 « ~§

0.05 .

 

 

 

0.“) I I I I I
0.00 0.02 0.04 0.06 0.08 0.10 0.12

Bulk Concentration (mM)

 

Figure 5.7: Fraction of proteins (r1) that desorb at an apparent rate of k1, as a function of
bulk protein concentration. Each data point represents the average of twenty experiments
conducted at room temperature (~20°C) and in the dark. The photobleaching pulse
duration was 100 ms.

44

1.0

 

0.9 J

0.8 a

0.7 ~

0.6 -
0.4 4

0.3 -

 

0.2 a

0.1 -

 

 

0.0 T I I I I
0.00 0.02 0.04 0.06 0.08 0.10 0.12

Bulk Concentration (mM)

 

Figure 5.8: Fraction of proteins (r2) that are more tightly held at the interface and desorb
at an apparent rate of k; as a function of bulk protein concentration. Each data point
represents the average of twenty experiments conducted at room temperature (~20°C) and
in the dark. The photobleaching pulse duration was 100 ms.

45

Figure 5.9 and Figure 5.10 show theoretical fluorescence recovery curves based on the
kinetic parameters obtained via curve fitting of experimental data to Eqn. [19] (see
Chapter 4), at a bulk concentration of 0.07 mM. The parameters used are in the third row
of data in Table 5.1 and Table 5.2. The dashed line in Figure 5.9 is qualitatively
comparable to the experimental data shown in Figure 5.1, and the dashed line in Figure
5.10 is qualitatively comparable to the experimental data shown in Figure 5.2. Note that
Figure 5.1 and Figure 5.2 represent data fi'om single experimental runs, whereas the
dashed lines in Figure 5.9 and Figure 5.10 were calculated fi'om parameters obtained
through the analysis of several experimental runs. Thus, we do not expect a quantitative

match between Figure 5.1 and Figure 5.9 or between Figure 5.2 and Figure 5.10.

The solid lines in Figure 5.9 and Figure 5.10 represent theoretical calculations of what the
experimental data would look like if slow photobleaching due to the monitoring beam did

not occur. These lines were calculated by setting k pb equal to zero in Eqn. [19]. As

Figure 5.9 shows, the effect of slow photobleaching is noticeable but not significant during
the first minute of fluorescence recovery. However, as is evident from Figure 5.10, the
effects of slow photobleaching become very significant over longer experimental runs. In
fact, the fluorescence level (dashed line) begins to drop at about 160 seconds. This has
profound implications for TIRF/FPR experiments mn over long times, as will be discussed
further in section 5.2. The immobile fraction [r0] is the most drastically affected
parameter. At a bulk protein concentration of 0.07 mM, r0 was found to be 0.78 when
slow photobleaching effects are neglected, compared to the actual r0 value of 0.46 i 0.04

obtained in this study. This is discussed in firrther detail in Chapter 6.

46

 

 

 

 

 

 

 

 

 

 

0.95 -

O
o
C
§ 0.9 -
2
8
u—_ 0.85 1
‘U
.8
'5 0.8 -
E
20 0 75

' A — Fluorescence recovery w ithout slow photobleaching

------- Fhorescence recovery w 'th slow photobleaching
0.7 . fl . . .
0 10 20 30 40 50 60

Time (s)

 

 

 

Figure 5.9: Short-term theoretical recovery curves generated from Eqn. [19], using the
kinetic parameters obtained via curve fitting. The bulk concentration is 0.07 mM. The
curve that assumes absence of slow photobleaching was generated by setting k p, equal to

zero in Eqn. [19].

47

 

 

0.95 ~
0.9 -
0.85 -
0.8 a
0.75 -
0.7 -
0.65 -

0.6 J

o 55 _ — Fluorescence recovery w ithout slow photobleaching
. ------- Fluorescence recovery w in slow photobleach'ng

0.5 r . .
0 200 400 600 800

Time (s)

 

 

 

Normalized Fluorescence

 

 

 

 

 

 

 

 

 

 

Figure 5.10: Long-term theoretical recovery curves generated from Eqn. [19], using the
kinetic parameters obtained via curve fitting. The bulk concentration is 0.07 mM. The
curve that assumes absence of slow photobleaching was generated by setting k p, equal to

zero in Eqn. [19].

48

5.2 Discussion

We have shown that Slow photobleaching due to a continuously impinging monitoring
beam can significantly influence fluorescence recovery data in TIRF/FPR experiments.
We were able to account for this effect by estimating a slow photobleaching rate constant

k W. There are two methods of obtaining k Pb: a) we can conduct experiments in which

the monitoring beam is allowed to continuously excite fluorophores at and near the oil-
water interface and fit the data to Eqn. [18], or b) we can run TIRF/FPR experiments and

fit the data to Eqn. [19]. Within experimental uncertainty, the values obtained for k P,

with these two methods were identical, suggesting that protein adsorption/desorption is

not coupled to slow photobleaching.

It is desirable to minimize the effects of slow photobleaching in experiments that run over
long periods of time. For example, a study of protein adsorption onto a clean (protein
free) interface would require monitoring the increase in fluorescence over a long period of
time as the protein solution is pumped into the experimental cell to slowly displace buffer
solution already in the cell. Unfortunately, we cannot use Eqns. [18] and [19] to account
for the effects of slow photobleaching in systems with flow, because the equations apply
only to systems in which there is no forced convection at or near the interface. Since we
cannot mathematically account for slow photobleaching effects in systems with flow, we

must explore methods to minimize the rate of slow photobleaching in such systems.

49

Slow photobleaching of fluorophores inevitably occurs when the monitoring beam strikes
the interface, with the rate of photobleaching depending mainly on the beam intensity for a
given bulk concentration. Reducing the monitoring beam intensity is often not a viable
solution because the signal to noise ratio decreases. Alternatively, an optical chopper can
be used during lengthy experiments to reduce the rate of unintended photobleaching. If a
chopper does not sufficiently reduce the rate of photobleaching, a timed shutter system
can be employed in which the monitoring beam is allowed to intermittently strike the
interface. A trade-off must then be made between the percentage of experimental time
that the monitor beam strikes the interface (i.e. the percentage of acquired data that is

relevant) and the rate at which slow photobleaching occurs.

For lengthy experiments in which a slow, monotonic increase in fluorescence is expected,
the shuttering method can be used. However, for experiments such as TIRF/FPR, the
shuttering technique would be more difficult to implement. In a TIRF/FPR experiment,
capturing the initial data points immediately following the photobleaching flash is crucial
to obtaining an appropriate value for In, because the rapid initial recovery described by k1
is only observable in the first few seconds after photobleaching. A poorly timed shutter

could cause these initial data points to be missed.

By using BSA-FITC bulk concentrations greater than 0.015 mM, we were reasonably
assured that bulk diffusional processes would not contribute significantly to fluorescence
recovery. However, lateral diffusion of interface-adsorbed proteins could still contribute

to the recovery. We circumvented this problem by using a photobleaching beam whose

50

diameter is approximately three times that of the monitoring beam. Jauhari (1997) showed
that, given the beam width and this ratio of diameters used in our apparatus, lateral
diffusion of unbleached surface-bound fluorophores into the field of view of the
monitoring beam occurs in about 900 seconds, which is much longer than the duration of

our TIRF/F PR experiments.

In addition, once the microscope objective is properly focused at the interface, it sees only
a small central portion of the monitoring beam spot. This may be confirmed by removing
the photomultiplier tube and visually observing the elliptical spot made by the monitoring
beam before and after proper objective focusing is achieved. When the microscope
objective is not focused on the interface, the elliptical spot made by the monitoring beam is
seen to cover only a small portion of the objective’s field of view. As the focus gets closer
to the interface, the elliptical spot covers more of the objective’s field of view until, finally,
only the central part of the spot can be seen as a proper focus is achieved. The time for
unbleached surface-adsorbed proteins to laterally diffuse into the field of view of the

microscope objective will then take even longer than 900 seconds.

5.3 Possible sources of error

We encountered noise and variability in the fluorescence recovery curves obtained in the
TIRF/FPR experiments. The values of k, and k2 appeared to be the most affected, as their
relatively large errors of estimation would suggest (see Table 5.1, Figure 5.3, and Figure

5.4). A possible explanation is that the system was not given sufficient equilibration time

51

between experimental runs. J auhari (1997) showed that, for the experimental apparatus
used in our study, BSA-FITC reaches equilibrium approximately five minutes afier the
proteins have been pumped into the experimental cell. We allowed the system to
equilibrate for at least 10 minutes after pumping stopped, so we do not believe insufficient

equilibration time is a significant issue.

While inconsistent microscope alignment and focusing could hinder reproducibility, the
monitoring beam has been found to maintain proper alignment for long periods of time (on
the order of days), as has a proper focus of the objective at the oil-water interface. The
use of a vibration isolation table to dampen vibrations from external sources helps
maintain the integrity and stability of the oil-water interface, which in turn helps maintain
the alignment and focus of the microscope objective. We can thus eliminate these factors

as significant sources of error.

Internal noise from the PMT accounts for less than 5% of the fluorescence signal in typical
TIRF/FPR experiments, and this background noise is stable over long periods of time, so

it can also be eliminated as a significant source of error.

At the present time, the signal to noise ratio of our system is not high enough to
distinguish minor factors such as rotational difi‘usion and structural unfolding of proteins
as they adsorb to the interface. It is possible that with improvements in the signal to noise
ratio, additional physically identifiable parameters could be added to the kinetic model,

which would potentially improve the model’s ability to repeatably predict experimental

52

data. At the current noise level of our experimental system, a biexponential kinetic model
gives the best fit to TIRF/F PR data without overspecifying the system. This study has

added the slow photobleaching rate constant, k pl, , to the biexponential kinetic model to

improve the model’s predictive ability.

53

6. SUMMARY AND CONCLUSIONS

We have established that a biexponential kinetic model fits TIRF/FPR data well and
provides an acceptable framework for describing the desorption kinetics of BSA-FITC at
the oil-water interface. Using two desorption rate constants, In and k2, allows us to
describe two types of desorbing proteins: k1 is the apparent desorption rate of proteins
loosely adsorbed at the interface, and k2 is the apparent desorption rate of proteins
adsorbed tightly at the oil-water interface. At a bulk protein concentration of 0.07 mM, k1
was estimated to be 0.52 i 0.12 5", while k2 had a value of 0.008 : 0.003 s". The noise
and variability of the fluorescence recovery curves obtained in this study suggests that
putting additional adjustable parameters into our current model would not add significant

usefirl information about the system behavior.

Conducting experiments in the reaction limited regime assured us that bulk diffusional
effects would not significantly affect the apparent rates of adsorption and desorption.
Burghardt and Axelrod (1981) showed that, for the glass-water interface, bringing the
bulk BSA-FITC concentration above 0.015 mM guarantees that the system is in the
reaction limited regime. We assumed that a similar transition into the reaction limited

regime would occur at the same concentration for the oil-water interface.

Figure 5.3 and Figure 5.4 provide evidence that a transition does indeed occur between
bulk concentrations of 0.0] mM and 0.04 mM. Above 0.04 mM k1 and k2 reach

reasonably stable levels that are no longer strongly dependent on concentration,

54

suggesting that bulk diffusional effects are not limiting the adsorption and desorption
processes. Experimental uncertainty in the values of k1 leaves open the possibility that the
transition into the reaction limited regime could occur at a bulk concentration higher than
0.04 mM (see Figure 5.3). However, Figure 5.4 shows an abrupt transition between 0.01
mM and 0.04 mM as the value of k2 jumps from 0.0015 1- 0.0004 s'1 to 0.008 t 0.002 s"
before stabilizing at that level. We conclude, therefore, that the transition between the
bulk diffusion limited and reaction limited regimes for BSA-FITC at the oil-water interface

occurs at approximately the same bulk concentration as at the glass-water interface.

Slow photobleaching due to the monitoring beam was found to have a significant effect on
the fluorescence recovery curves obtained in our study. By adding the slow

photobleaching rate constant k pb to our mathematical model, we were able to successfirlly
account for this factor. Two independent methods of determining k P, (Eqns. [18] and

[19]) yielded identical values, within experimental uncertainty.

As Figure 5.10 illustrates, slow photobleaching dominates the system behavior over long
experimental periods. Initially, fluorescence increases due to the replacement of
photobleached fluorophores at the interface by unbleached fluorophores from the bulk. As
the rate of fluorescence recovery slows down over time, slow photobleaching causes the
fluorescence signal to drop. In order to determine the effect of slow photobleaching on
the kinetic parameters obtained in our study, we fit Eqn. [15] to the recovery curve
predicted by Eqn. [19] at a bulk concentration of 0.07 mM (shown in Figure 5.9 as the
dashed line).

55

The magnitudes of k1 and kg were almost unchanged, as k1 was found to be 0.51 s'1 and kg
was found to be 0.009 5", compared to 0.52 i 0.12 s'1 and 0.008 i 0.003 s", the actual
values obtained for a bulk concentration of 0.07 mM. The fraction of proteins which are
loosely adsorbed at the interface, r1, also remained almost unchanged, with a value of
0.09. However, both r0 and r2 changed drastically: r0, the immobile fraction, was
calculated to be 0.78 and r; was found to be 0.13, compared to 0.46 i 0.04 and 0.45 i

0.05, the actual values obtained for a bulk concentration of 0.07 mM.

We conclude from this that the main consequence of neglecting the effects of slow
photobleaching is to overestimate the immobile fiaction r0. Note that only the first 60
seconds of data were used in this exercise. Curve fits involving data from longer
experiments in which slow photobleaching is more significant would most likely produce
much larger errors in the values of k1 and k; in addition to overestimating the immobile

fraction.

56

7. SUGGESTIONS FOR FUTURE WORK

The goal of this thesis was to quantify the desorption kinetics of BSA-FITC at the oil-
water interface. It serves as an important step towards using total internal reflection
fluorescence to study more complex phenomena such as receptor-ligand interactions at the
oil-water interface. Jauhari (1997) used the TIRF/FPR technique to study the surface and
bulk diffusion coefficients of BSA-FITC at the oil-water interface, using the same basic
experimental apparatus used in this study. This thesis builds on Jauhari’s work by
expanding the utility of TIRF/FPR as a tool to quantify the behavior of proteins at the oil-
water interface. Modifications to our experimental setup are ongoing. A 70° dovetail
prism will replace the 64° prism currently used. This will decrease the evanescent wave
penetration depth (see Eqn. [3]), making the system more surface-selective. After this and
other pending modifications are completed, we suggest that the following studies be

conducted.

7.1 Protein adsorption onto a clean interface

By slowly infirsing a protein solution into the experimental cell of our TIRF/FPR
apparatus (filled initially with the buffer used to make the protein solution) and monitoring
the increase in fluorescence over time, quantitative information could be obtained about
the kinetics of protein adsorption onto a clean oil-water interface. As is usual in such
experiments, we would expect to see an initial flat fluorescence baseline, which would be

characteristic of the “dead time” between the start of protein infiision and the arrival of

57

proteins into the field of view of the monitoring beam. The fluorescence signal would then
increase steadily over time as protein molecules adsorb onto the interface. As the
interface approaches saturation, there will be a decline in the rate of change of the
fluorescence signal, and finally, the signal level will stabilize as the system reaches

equilibrium.

Parameters describing the characteristics of the system, such as the dead time and the
equilibration time, could be obtained. Several protein infirsion rates could be used, and the
change in fluorescence with time could be monitored for each infirsion rate. These data
would be useful in constructing a model for adsorption onto a clean interface that
incorporates the effects of fluid flow. Infusion experiments could be conducted at several
protein concentrations to yield information about the efi‘ect of concentration on the

equilibration time.

The following precautions should be taken when conducting these experiments. The
critical infusion rate at which shearing effects disrupt the integrity of the oil-water
interface must not be exceeded. Jauhari (1997) reported this rate to be 0.17 mL/min for
our apparatus. Slow photobleaching, which has been shown to be especially significant in
lengthy experiments, would have to be dealt with. The most efl‘ective way to do this
would be to employ a system in which the monitoring beam is shuttered so that it only
strikes the interface periodically, during periods when data are being acquired. The
optimum times for exposing and shuttering the monitoring beam could be determined

experimentally. Criteria for this optimization are that slow photobleaching should be

58

minimized, while important trends in the data (such as the abrupt fluorescence increase

when fluorophores reach the interface) must not be lost due to beam shuttering.

An additional concern regarding this study is that we are somewhat unsure about what
fraction of the fluorescence signal comes from bulk proteins. Thus, it would be difficult to
obtain an adsorption rate constant fiom these experiments, because part of the increase in
fluorescence signal over time would come from the increase in bulk protein concentration
during the infirsion process. Currently, there is not a reliable technique for determining the

surface concentration of proteins at the oil-water interface for a given bulk concentration.

The development of such a technique would enable us to calculate the ratio of bulk
fluorescence to interfacial fluorescence, and knowing this ratio would enable us to include
bulk diffusional effects in the determination of an adsorption rate constant. This ratio is
given by Zd/C , where Z is the bulk concentration, C is the surface concentration
(which we cannot presently determine), and d is the evanescent wave penetration depth,
which is known. Burghardt and Axelrod (1981) reported that this ratio is 0.07 for the
highest BSA-FITC concentration used in their study (techniques are readily available for
determining surface concentrations at the solid-liquid interface), implying that at least 93%
of the observed fluorescence is from surface adsorbed proteins. Until we can determine
the surface protein concentration for a given bulk concentration, we cannot readily
calculate an adsorption rate constant using protein infusion experiments. However, other
useful information, such as the dependence of the dead time and the equilibration time on

bulk concentration and infusion rate, can be readily detemrined.

59

7.2 Effect of labeling ratio on protein desorption kinetics

To study the effects of fluorescent labels on the behavior of proteins at the oil-water
interface, the study conducted in this thesis could be expanded upon by conducting
TIRF/FPR experiments at multiple labeling ratios. Robeson (1995) has reported that, as
the labeling ratio is increased, elevated levels of concentration quenching accelerate the
apparent recovery kinetics and cause the immobile fraction to be underestimated. As a
result, concentration quenching would clearly have to be taken into account. The simplest
way to circumvent the problem of concentration quenching is to use labeling ratios for
which concentration quenching efi‘ects are not present. For BSA-FITC, concentration
quenching was found to be absent for labeling ratios between 0.03-0.80 (Robeson 1995).
For higher labeling ratios, Robeson (1995) has developed an analysis technique that takes

into account the effects of concentration quenching on fluorescence recovery curves.

60

8. BIBLIOGRAPHY

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62

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