Journal o f Biochemical and Biophysical Methods, 1 (1979) 195--208
© Elsevier. North-Holland Biomedical Press
195
THE MEASUREMENT OF SUBNANOSECOND FLUORESCENCE DECAY
OF FLAVINS USING TIME-CORRELATED PHOTON COUNTING AND A
MODE-LOCKED Ar ION LASER
A.J.W.G. VISSER 1 and A. VAN HOEK 2
1 Department o f Biochemistry, Agricultural University, De Dreijen 11, 6703 BC
Wageningen, and 2 Laboratory o f Molecular Physics, Agricultural University,
De Dreijen 6, 6700 EP Wageningen, The Netherlands
(Received 8 March 1979; accepted 27 March 1979)
A system is described consisting of a mode-locked Ar ion laser and time-resolved
photon-counting electronics. The system is capable of measuring fluorescence lifetimes in
the subnanosecond time domain. The Ar ion laser is suitable for the excitation of flavins,
since the available laser wavelengths encompass the first absorption band of the yellow
chromophore. Due to the high radiation density and the short pulse, both the time and
wavelength resolution of the fluorescence of very weakly emitting compounds can be
measured. Experiments have been described for flavin models exhibiting single and
multiple modes of decay. In these examples lifetimes were determined both from
deconvolved decay curves and from direct analysis of the tail of the curve, where no
interference of the exciting pulse is encountered. Both determinations showed very good
agreement. Due to the highly polarized laser light the decay of the emission anisotropy
could be measured directly after the exciting pulse. In principle, fast rotational motions
might be detected. An anisotropy measurement conducted with a flavoprotein with a
noncovalently attached FAD is presented.
Key words: mode-locked laser; flavins; fluorescence decay; emission anisotropy.
INTRODUCTION
T h e f l u o r e s c e n c e of flavins is s t r o n g l y i n f l u e n c e d b y i n t e r a c t i o n s w i t h
a r o m a t i c o r e l e c t r o n - r i c h m o l e c u l e s , w h i c h are in close p r o x i m i t y t o t h e
y e l l o w c h r o m o p h o r e . T h e d y n a m i c p a r t o f t h e s e i n t e r a c t i o n s gives rise n o t
o n l y t o a d e c r e a s e o f t h e f l u o r e s c e n c e e f f i c i e n c y , b u t also t o a d r a s t i c s h o r t e n ing o f t h e f l u o r e s c e n c e l i f e t i m e [ 1 - - 3 ] . W h e n t h e flavin is o n l y s u r r o u n d e d
b y s o l v e n t m o l e c u l e s t h e m e a s u r e d l i f e t i m e s are in o r d e r o f 4 - - 1 0 ns d e p e n d ing o n t h e c o n d i t i o n s e m p l o y e d [ 4 ] . T h e s e r a t h e r l o n g l i f e t i m e s c a n e a s i l y
be d e t e r m i n e d due to the high q u a n t u m yield of the fluorescence. On the
o t h e r h a n d , t h e f l u o r e s c e n c e p e r t u r b e d b y i n t e r a c t i n g g r o u p s h a s l o s t conAbbreviations: FWHM, full width at half-maximum; CW, continuous wave; MCA, multichannel analyzeR; PMT, photomultiplier; TAC, time-to-amplitude converter.
196
siderable intensity and is characterized by time constants sometimes in the
subnanosecond
time domain
[21. In order to obtain accurate dynamic
information we used a mode locked Ar ion laser as excitation source. This
typed laser provides short pulses (FWHM
less than I00 ps) of high radiation
energy, at wavelengths in the blue spectral region. This is in perfect coincidence with the red portion of the lowest absorption band of the flavin. In
this paper the results of the decay measurements
are presented that were
conducted with suitable model compounds, namely 3,7,8,9,10-pentamethylisoalloxazine in water with and without the presence of the collisional
quencher potassium iodide and a flavinyltryptophan peptide [4,5]. The
virtually I00% linearly (vertically) polarized laser light permits one to obtain
the decay of the emission anisotropy directly after the pulse from which in
principle rapid rotational motion of the bound flavin in flavoproteins can be
monitored. Experiments of this type are described with the flavin bound as
eofactor FAD to the enzyme lipoamide dehydrogenase [6].
MATERIALS
AND
EQUIPMENT
Materials
3,7,8,9,t0-Pentamethylisoalloxazine was synthesized according to Grande
et al. [7]. The N(3)-substituted flavinyltryptophan methyl ester peptide was
prepared according to FSry et al. [SJ. Lipoamide dehydrogenase (EC
1.6.4.3) was purified from pig heart as described by Massey et al. [9].
Erythrosine B was purchased from Eastman Kodak and was used as received.
All reagents used were of analytical grade and buffers were made from
doubly distilled water.
I n s t r u m e n ts
Absorption spectra were obtained with a Cary 14 spectrophotometer and
fluorescence spectra were recorded on an Aminco SPF-500 spectrofluorometer. The laser system and associated optical and electronical components are
schematically depicted in Fig. I. The laser is a Coherent Radiation Model CR
18 UV Ar ion laser. The mode-locking acousto-optic element is a brewster
prism (Harris Corp.), driven by a 38 070 MHz signal from the frequency
synthesizer ND 60 M from Schomandl. The different lines at 514.4, 501.7,
496.5,476.7,472.7
and 457.9 nm can be mode locked individually. In the
present study the 476.7 nm line was used. The 76 140 MHz optical pulses
were monitored with a fast photodiode and a sampling oscilloscope (Hewlett
Packard 1430C, 1811A, 181A). The measured total FWHM
was about 180
ps. It c a n be expected, that the FWHM o f the light pulse is less ~han l O O p s
when corrected for the rise time of the detector. At 25 A laser discharge
current the o u t p u t of the laser was about 400 mW (CW) and about 100 mW
of averaged mode locked power. With beam splitters about 0.5--2 mW of
197
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i- 2-. h
-I
s+;____._I_, _p__+ 59
:
s
-*$o
I
I
I
-j___
-’
’
L -d
I
Fig. 1. Block diagram of theexperimental
set-up. 1, frequency synthesizer; 2, variable gain
amplifier; 3, acoustooptic
modulator; 4. Ar ion laser; 5, power meter; 6, beam splitter; 7,
variable density filter; 6, optical polarizer; 9, sample cuvet; 10, cut-off filter; 11, monochromator or interference
filter; 12. photomultiplier;
13, fast amplifier; 14, constant
fraction discriminator; 15, time-to-amplitude
converter; 16, 100 MHz discriminator; 17,
variable time delay; 18, analog to digital converter; 19,multichannel
analyzer; 20, cassette
tape deck; 21, computer; 22, sampling oscilloscope; 23, fast photodiode; 24, biajPsupply.
averaged mode locked power were deflected towards the sample compartment. The detection photomultiplier (PMT) is a Philips XP2020 placed in
a thermoelectric-cooled
housing of Products of Research Inc. in order to
reduce background noise. The electronical part consists of modules of the
Ortec 9200 system and of a Laben model 8001 multichannel analyzer
(MCA). The time scale was calibrated by an Ortec model 462 time calibrator.
Because of the very inefficient use of information when the time-to-amplitude converter (TAC) is started with the very high frequency of the laser
light pulses, the siart and stop channels were reversed [lo] (dead time of the
TAC is 5 ~1s). The TAC was started with an emission photon pulse and
stopped with a trigger pulse, initiated by the 38 070 MHz signal of the
synthesizer. Scattering from glycogen solutions and fluorescence data were
collected at a constant starting rate of about 25 kHz. For the scatter solution
the exciting beam was additionally attenuated to l-2 PW averaged modelocked power with neutral density filters (Balkers). To eliminate effects of
rotational motion on the lifetimes, a polarizer (Polacoat) was placed at 55”
with respect to the vertical in front of the monochromator
(Jarrell Ash
82410, band width 6 nm) in the detection light path [ 111.
The polarizers used for the emission anisotropy measurements were Glan
Thompson prisms. The polarized emission components (i,,(t) and i*(t)) were
viewed through a Schott KV 520 cut-off filter and a Balzers 554 nm interference filter. The polarizer in the detection light path is clamped in a motordriven rotatable
mount
and can be rotated over 90” and back by applying
198
logic pulses to an activating relay. By using the timer of the MCA for supply
of the logic pulses it is possible to measure ill(t) and i±(t) alternately during a
preset time and to store the results into two m e m o r y subgroups. The
instrumental response function was obtained in the same way from a diluted
glycogen scattering solution replacing the sample under investigation. The
filters in the detection light path were removed. Since the scattered light
from glycogen solutions is highly polarized (>95%) only i!l(t ) of the scatter
solution was employed in subsequent deconvolution procedures.
METHODOLOGY
Data analysis
In order to account for the finite duration of the exciting pulse in the
analysis of the complete decay curve one has to rely on deconvolution
procedures. We used the nonlinear least-squares method for deconvolution
of the fluorescence decay [12]. Some
data were also analyzed with a
response function (g(t)) obtained from a quantum counter in order to check
the wavelength dependence
of the detector [13]. A suitable quantum
counter for the flavin system is erythrosine B in water since it has a very short
lifetime of about 90 ps [14].
Polarized decay data were analyzed as described by Dale etal. [15].
Multiplication factors in order to correct for imperfect polarizers were
obtained as follows [!5,16]. The decay profiles of ill(t) and i~(t) of a fluorescent solution were semi-simultaneously collected in two different halves of
the MCA
memory. After correcting for background, the integrals of ill(t) and
i±(t) over N channels were determined. In a separate experiment the steadystate degree of polarization (p) was accurately measured [6]. All subsequent
curves (i±(t)) were multiplied with the following normalization factor:
T
il¢(t) dt
N o - 1 --p o
l+p
T
f
(I)
il(t) dt
0
where T is the last point of the decay curve in the MCA.
Sum and difference decays, determined from ill(t ) and il(t), can be considered as convolution products:
t
s(t) = A,(0 + 2iz(t) = / g(t -- t')S(t') dr'
0
(2)
t
d(t) = ijj(t) -- i±(t) = f g(t -- t')D(t') dt'
0
199
Whenever necessary the total fluorescence s(t) can be analyzed in a sum of
exponential terms:
S(t) = ~
~i e-~/~
(3)
i=l
the parameters were obtained after a search for the minimum value of X2 (cf.
legend of Fig. 3).
The anisotropy decay r(t) follows from
r(t) -
d(t)
(4)
s(t)
Usually r(t) is written as
&
r(t) = 2_J ~i e-t/¢i
(5)
/=1
where n is equal to either 1 or 2, ¢i is the correlation time for the decay of
the anistotropy, ~i the preexponential factor and E[~=l~i the anisotropy at
time zero. As pointed out by Dale et al., r(t) can be obtained from
deconvolution of the difference decay according to two models [15]:
(1) The multiexponential character of s(t) arises from microheterogeneity
of the fluorophore (different environments giving rise to different lifetimes)
in which the difference decay is analyzed from
d(t) = ~
ri(t)si(t)
(6)
i=l
where i indicates the individual species.
(2) There exists rotational anisotropy in a single environment from which
d(t) is c o m p u t e d as the following convolution product:
n
d(t) = r(t) X S(t) = g(t) X ( ~
i=1
tn
(Ji e t / ~ i ) ( ~ ~i e-t/ri)
(7)
i=I
In principle the decay of the anisotropy can be derived from the sum and
difference decay curves (Eqns. 2 and 4) and analysis can be performed
directly w i t h o u t reference to t h e two models.
Ern ission spectra
In Fig. 2 normalized spectra are shown from the compounds used in this
work. The wavelengths of excitation and emission employed in the decay
experiments as well as that of the expected Raman line of water are also
indicated in the figure.
200
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4
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2
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w
u
4
3
2
w
o
tL
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HAVELENGTH ( N M )
Fig. 2. Peak normalized emission spectra of the compounds used in this work, Exeitation
wavelength was 476 nm. Optical densities were adjusted to 0.05 at 476 nm. (1) 3,7,8,9,10-pentamethylisoalloxazine in 50 mM s o d i u m phosphate, pH 7.0; (2) erythrosine B in
50 mM s o d i u m phosphate pH 7.0; (3) N(3)-flavinyltryptophan m e t h y l ester in 50 mM
s o d i u m phosphate pH 7.0; (4) lipoamide dehydrogenase in 30 mM sodium phosphate/0.3
mM EDTA pH 7.2. Starting from the shortest wavelength the four arrows indicate respectively the exciting wavelength (4,76 nm), the emission wavelength (530 nm) in ease of the
peptide, the observation wavelengths (550 and 554 nm) for the m o d e l flavin, erythrosine
B and lipoamide dehydrogenase and the expected Raman line of water (568.9 n m ) w h e n
exeitation is at 476.7 nm.
Examples
As an example of the range of lifetimes that can be measured the decay
profiles of aqueous solutions o f scatter (glycogen), erythrosine B and
3,7,8,9,10-pentamethylisoalloxazine (quenched with KI) are presented in
Figs. 3A and B° The convoluted curves and the deviation functions of the
residuals are also s h o w n *. The single p h o t o n data were collected during
a fixed period o f a multiple of 100 s to accumulate at least 40 000 counts in
the maximum. Background collected during the same period of time was
subtracted from the decay curve. The scattered laser light pulse as seen by
t h e detection system has a FWHM of 5 0 0 - - 6 0 0 ps. The reason is that the
excitation pulse is broadened in time by transit time variations in the PMT
and broadened and somewhat distorted by time w a l k in the constant
fraction-timing discriminator. In case of u n q u e n c h e d or partly quenched
* The exact nature o f the oscillation, w h i c h becomes manifest in the deviation curve, is
not k n o w n . The oscillation spoils the X2 value, but does not appear to effect the reproducibility of the parameter values. The standard deviation of the parameters was always
less than the m o s t significant digit in the reported values.
I ~11
i
1
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=_=
i
2
I
-
i
3
TIME (NS)
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i
4
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i
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TIME "NS)
,~
I _
7
g
~
CH 3 CIH3
0
;
B
10
N
N i=1
Xa = 1 ~
(F(i) - - Fc(i)) 2 .
F(i)
N = n u m b e r o f c h a n n e l s used in t h e MCA. A, e r y t h r o s i n e B in 5 0 m M s o d i u m p h o s p h a t e p H 7 . 0 ; 7 = 8 0 . 2 ps; X 2 = 11. B, 3 , 7 , 8 , 9 , 1 0 p e n t a r n e t h y l i s o a l ] o x a z i n e in 5 0 m M s o d i u m p h o s p h a t e p H 7 . 0 q u e n c h e d w i t h 4 5 mM KI; ~- = 1.01 ns, X 2 = 64.
D V ( i ) = (F(i) - - F e ( i ) ) / N / ~ ;
Fig. 3. F l u o r e s c e n c e d e c a y curves o f t h e m o d e l c o m p o u n d s u s e d . E x c i t a t i o n profiles ( g ( t ) ) , e x p e r i m e n t a l ( F ( t ) ) and calculated ( F c ( t ) )
d e c a y curves are s h o w n . F c is t h e result o f a c o n v o l u t i o n o f g ( t ) w i t h a single e x p o n e n t i a l f u n c t i o n . T h e deviation f u n c t i o n ( D V ( i ) ) o f
t h e w e i g h t e d residuals in each c h a n n e l i is d i s p l a y e d o n t o p o f t h e d e c a y curves.
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202
flavin fluorescence, the tail of the decay curve can be analyzed directly without taking the finite width of the exciting pulse into account. The data
obtained from the rapidly decaying samples required deconvolution to
exgrac~ the correct time consCan~(s). The fluorescence of erythrosine B is
completely relaxed within the time lapse between two laser pulses, therefore
analysis has to be performed across the whole decay profile. In fact, the
fluorescence response appears as an exciting pulse profile a few channels
shifted in time with respecg go the impulse response (see Fig. 3A)o From
three independent measurements
the time constant of erythrosine B is
88 -+ I0 ps in accordance with the reported values of 90,110 and 57 ps (see
references in ref. i4). In orde~ to investigate the wavelength dependence of
the detector 3,7,8,9,10-pentamethylisoailoxazine
can be employed
as a
convenient standard having one single lifetime [4]. The data can be analyzed
using the following impulse response functions:
(i) The laser pulse measured with a scatter solution a¢ the exciting wavelength.
(2) The pulse determined at the emission wavelength using a compound
with a well determined single short lifetime (erythrosine B: T = 88 ps).
(3) The-pulse measured at the exciting wavelength, but purposely shifted
over a certain fraction of time [17].
We determined the lifetime of 3,7,8,9,10-pentamethylisoalloxazine
in
wager using the three different response functions in the convolution procedure. Neither a positive nor a negative shift in time of the excitation pulse
gives an improvement
of the quality of the fit. As judged from the deviation
function and the cons;an~ X 2 values it is to be noted that the first two
methods
all lead to the same mono-exponential
lifetimes. All results
obtained with the quenched
and unquenched
3,7,8,9,10-pentamethylisoalloxazine are collected in Table I.
TABLE 1
DYNAMIC FLUORESCENCE
LOXAZINE
QUENCHING
OF
3,7,8,9,10-PENTAMETHYLISOAL-
T h e c o m p o u n d ( 1 0 pM) was dissolved in 0.1 M s o d i u m p h o s p h a t e , p H 7.0 Q u e n c h e r (KI)
was a d d e d f r o m a 3 M s t o c k s o l u t i o n in water.
Concentration
of quencher
(mM)
~a
(ns)
TO/T
F 0/F b
0.0
45
150
1:72 ( 1 . 7 3 )
1.01 ( 1 . 0 0 )
0.52 ( 0 . 5 1 )
1.00
1.73
3.39
1.00
2.04
5.14
a Results f r o m a o n e c o m p o n e n t analysis. T h e values b e t w e e n b r a c k e t s were o b t a i n e d
f r o m analysis o f t h e tail o f t h e d e c a y curve s t a r t i n g at a b o u t 2.5 ns a f t e r t h e e x c i t a t i o n
m a x i m u m (cf. Fig. 3B).
b Fluorescence intensity ratio obtained by comparing peak maxima.
I I~1
I @2
1 04
I ~6 |
@
-51 ~ t
1
I
2
~
I
I
CH 3
I
3
S
"~
6
o
_1
TIME (NS)
II I
H3C.x.~-..~N.x~,,fN_
I
nil
0
I
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H
8
A~
9
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3
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Z
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k.<
i 03
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1 @6
-13
@
@
1f
1
2~
3
~
TIHE
5
(NS)
6
7-
B
9
1 ~
Fig. 4. Fluorescence d e c a y curve o f the f l a v i n y l t r y p t o p h a n p e p t i d e in 50 mM s o d i u m p h o s p h a t e pH 7.0. Details as described in the
legend o f Fig. 3. A , d o u b l e e x p o n e n t i a l fit o f the e x p e r i m e n t a l data; (~t = 0.92; T1 = 0 . 3 3 ns; ~2 = 0.08; T2 = 3 . 7 0 ns; X 2 = 2 0 0 . B, single
exponential fit o f the tail o f the decay curve; 1"1 = 3 . 8 0 ns; X2 = 23 ; t h e c o n v o l u t e d curve is p l o t t e d over the entire region.
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204
A n o t h e r example of a flavin c o m p o u n d exhibiting a very short fluorescence lifetime is given by t h e f l a v i n y l t r y p t o p h a n peptide. Because of the
close p r o x i m i t y o f the ~ryptophan residue the fIavin fluorescence in the
intramolecular complex is strongly quenched. T he experimental results obtained with this peptide are presented in Figs. 4A and 4B. The best fit is
obtained b y simulating the experimental decay with a biexponentia! decay
function, In this particular case a long lifetime c o m p o n e n t (3.7 ns) was
present, Because of the short laser tight-pulse repetition time (12.8 ns) a tail
o f the fluorescence response is present a~ the time that a new fluorescence
event is created. This tail is the result of the response on the previous
impulse and contributes ~o the total decay, One m e t h o d to avoid this
p r o b lem is to put an exponential term with the longes~ lifetime through the
tail and subtract this c o n t r i b u t i o n from the total decay curve. In this
example it tu r ne d out that analysis w~th and w i t h o u t subtraction yielded
nearly the same parameter values. The het erogenei t y is probabl y due to the
presence o f different conformers in dynamic equilibrium. The fluorescence
o f the free flavin is m o n o e x p o n e n t i a l and has a value of 4.8 ns [4]. It should
13
T
w
D
-13
I
I
I
I
r
I
f
[
4
5
TIME (NS)
6
0.~
0.3
>n
b-
ot ~ 0.2
<
0.1
J
I
2
3
Fig. 5. D e c a y o f emission a n i s o t r o p y o f F A D in l i p o a m i d e d e h y d r o g e n a s e . The n o i s y
c u r v e i s t h e e x p e r i m e n t a l m ~ i s o t r o p y a n d t h e straigh~ i i h e r e f l e c t s t h ~ ~ a ~ d u l a f 4 d - t F m e
d e p e n d e n c e o f t h e a n i s o t r o p y w i t h a single c o r r e l a t i o n t i m e o f 25.7 ns; X2 = 6.6 • 10 -4.
T h e deviation f u c t i o n is m u l t i p l i e d b y 104. T h e t e m p e r a t u r e was 4 0 . 0 ° C and t h e c o n c e n t r a t i o n o f e n z y m e was 1 m g / m l in 30 m M s o d i u m p h o s p h a t e / 0 . 3 m M E D T A p H 7.2.
205
be n o t e d t h a t analysis in two exponential terms leads to values less than 4.6
ns.
Decay o f the emission anisotropy
E r y t h r o s i n e B emission is strongly polarized due to the very short lifetime
o f the fluorescence. The flavin fluorescence in t he dimeric flavoprotein
lipoamide dehydrogenase is polarized as well, but the reason for this is t hat
t h e flavin is b o u n d rigidly t o the protein, which rotates on a much slower
time scale. In Fig. 5 we present the experimental decay of the emission
anisotropy o f the flavin in lipoamide dehydrogenase. The onset is at t he
m a x i m u m of the fluorescence response. The experimental data can be fitted
with a single correlation time (~c = 25.7 ns) as is shown by the straight calculated curve and the deviation f u n c t i o n (Fig. 5). Analysis was also
p e r f o r m e d in terms of t he t w o models represented by equations 6 and 7.
These results and o t h e r available data are collected in Table 2, Because of
TABLE 2
ANISOTROPY CHARACTERISTICS OF LIPOAMIDE DEHYDROGENASE
T h e results o f d o u b l e e x p o n e n t i a l fits o f sum (S) a n d d i f f e r e n c e (D) decays (cf. Eqn. 3)
are also p r e s e n t e d . C o n d i t i o n s as given in t h e legend o f Fig. 5.
OQ a
T1 a
(ns)
~2 a
T2 a
(ns)
( T)b
(ns)
~l c
(ns)
~2 d
(ns)
~3 e
(ns)
44 f
(ns)
rO g
( r)h
S
0.51
0.49
0.49
2.03
1.72
25.7
26.0
25.2
(22.9)
27.1
0.338
0.326
D
0.52
0.51
0.48
1.91
1.61
a S y m b o l s refer to c o n v o l u t i o n o f t h e laser pulse with t h e d o u b l e e x p o n e n t i a l f u n c t i o n
2
2
i=l
(~ie - t [ ~ i , ~
OLi = 1.
i=1
2
2
b Average l i f e t i m e d e f i n e d as: ~
aiT~ / ~
a~'i.
i= l
i= l
c C o r r e l a t i o n t i m e o b t a i n e d f r o m o n e c o m p o n e n t analysis o f e x p e r i m e n t a l a n i s o t r o p y
decay.
d C o r r e l a t i o n t i m e d e t e r m i n e d f r o m t h e d i f f e r e n c e curve (D) a c c o r d i n g t o Eqn. 7 assuming a single t i m e c o n s t a n t for m o t i o n a n d t h e l i f e t i m e s a n d a m p l i t u d e s as d e t e r m i n e d
f r o m t h e t o t a l d e c a y S.
e C o r r e l a t i o n t i m e calculated f r o m 1/7~2 - - 1/~"s a n d (value b e t w e e n b r a c k e t s ) I [ ( T ) D - 1/( T)S.
f C o r r e l a t i o n t i m e f r o m t h e e m p i r i c a l f o r m u l a f o r g l o b u l a r p r o t e i n s [ 18 ]: 44 = ( M / 3 . 6 9 ) •
10 -3 ns w i t h M = 1 0 0 0 0 0 d a l t o n s .
g L i m i t i n g a n i s o t r o p y a t t i m e zero a f t e r t h e pulse (cf. Fig. 5).
h Steady-state emission anisotropy.
206
the short averaged fluorescence lifetime a single correlation time is calculated
but as judged from the ]ow X 2 value (Fig. 5) the single time constant
sufficiently reflects the motion of the bound flavin within the short observation time of 12 ns. The values in Table 2 axe very close, which means that in
this particular case discrimination between the two models is not possible.
Also included is the strictly empirical value of the rotational correlation time
for a sphere of comparable volume as for a globular protein with molecular
weight of 100 000 [18].
DISCUSSION
The system as outlined is suitable for lifetimes less than 1.7 ns, since at
least 99.5% of the fluorescent population is returned to the ground state
after 12.8 ns (i.e. the time lapse between two laser pulses). The use of the
full pulse train might lead to artifacts, especially when high radiation
energies are employed or when the lifetimes are too long [!4]. In the latter
case the fluorescence response on the previous impulse interferes with the
instantaneously excited fluorescence. Due to the improved time resolution
by the use of ps laser pulses~ the potential of measuring accurate biexponential decays is extended towards the subnanosecond
time region. This is
demonstrated in the experiment with the peptide, which exhibits a distinct
heterogeneity in the fluorescence kinetics. The results can be interpreted in
a way that more efficient dynamic quenching by ~ryptophan takes place in
the conformer characterized by the shortest lifetime (0.33 ns). The other
conformer (~ = 3.8 ns) corresponds with a more unfolded configuration. If in
a first approximation the amplitudes are related to concentration, about 92%
of the equilibrium mixture consists of the strongly quenched conformer,
whereas 8% exists in a more open form. Experiments of this kind have been
performed
for compounds
exhibiting much
longer lifetimes [19]. The
sensitivity of the instrumentation permits one to obtain decay curves from
solutions containing even less than 10 -1° M flavin. With these very diluted
samples one has to be aware of contributions of background and impurity
emission interfering with the fluorescence° An additional problem arising in
flavoproteins is that the contribution of flavin released from the protein
might become predominant in the fluorescence at these very low concentrations. A distinct advantage is that this system offers the possibility of direct
analysis of the emission anisotropy. Using flash-lamp excitation this mode of
analysis could only be applied successfully with the employment
of fluorescent probes with very long lifetimes like several pyrene derivatives [20]. In
the example of lipoamide dehydrogenase the FAD seems to be rigidly bound
to the protein, since the correlation time obtained from the time dependence
of the emission anisotropy is in good agreement with the correlation time for
a spherical protein. On the other hand, one should bear in mind that the
averaged fluorescence lifetime is only 1.7 ns. Such a value is rather short as
compared
to the much
slower rotational diffusion of the protein. In
207
p r i n c i p l e , w i t h t h e m e t h o d d e s c r i b e d o n e s h o u l d be a b l e t o m o n i t o r f a s t e r
m o t i o n s l o c a l i z e d a t t h e site o f a t t a c h m e n t o f t h e f l u o r o p h o r e .
We are c u r r e n t l y e x t e n d i n g t h e s y s t e m f o r m o r e v e r s a t i l e o p e r a t i o n b y
t h e a d d i t i o n o f a s y n c h r o n o u s l y p u m p e d d y e laser, a f r e q u e n c y d o u b l e r a n d
an e x t r a - c a v i t y e l e c t r o - o p t i c d e m o d u l a t o r as p u l s e p i c k e r . T h e d a t a collect i o n e f f i c i e n c y c a n b e d o u b l e d w h e n t h e s t o p c h a n n e l of ~he T A C is t r i g g e r e d
w i t h a 76 1 4 0 M H z signal, e.g. f r o m a n o t h e r f a s t p h o t o d i o d e . T h e s e e x t e n sions i m p r o v e t h e t i m e r e s o l u t i o n a n d e x p a n d t h e r a n g e o f b o t h e x c i t a t i o n
wavelengths and lifetimes.
SIMPLIFIED DESCRIPTION OF THE METHOD AND ITS APPLICATIONS
The time resolution and the sensitivity of the system used to obtain fluorescence lifetimes has been improved considerably by utilizing a mode-locked Ar ion laser as source of
excitation. The ability to make time-resolved fluorescence measurements is very useful in
the study of the biological and physical properties of flavins and flavoproteins as well as
many other fluorescent biological molecules. The advantage of this particular system is
that accurate decay curves o~ biological molecules with short fluorescent lifetimes and
weak emissions can be obtained during a short measuring time. The experimental decay
curves allow visual distinction between single and double exponential decay even in the
picosecond time region. Another advantage of the employment of mode-locked laser
systems coupled with fast detection electronics is that the decay of the emission anisotropy can be determined with good precision and time resolution. In this way (sub)nanosecond time constants for rotational motion of biological molecules can be obtained
directly from the experimental emission anisotropy.
ACKNOWLEDGEMENTS
We are i n d e b t e d t o Mr. D. B e b e l a a r f o r l o a n o f t h e f a s t p h o t o d i o d e a n d
f o r m a n y h e l p f u l s u g g e s t i o n s , t o Drs. F. Miiller, C. V e e g e r a n d W.H. S c o u t e n
f o r t h e i r i n t e r e s t , t o Mr. B.J. S a c h t e l e b e n f o r d r a w i n g t h e s t r u c t u r a l f o r m u l a e
a n d F i g . 1, a n d t o , Mrs. J.C. T o p p e n b e r g - F a n g f o r t y p i n g . This w o r k w a s supp o r t e d in p a r t b y t h e N e t h e r l a n d s F o u n d a t i o n f o r C h e m i c a l l~esearch
( S . O . N . ) w i t h f i n a n c i a l aid f r o m t h e N e t h e r l a n d s O r g a n i z a t i o n f o r t h e
A d v a n c e m e n t o f P u r e R e s e a r c h (Z.W.O.).
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