Fl uo r e s c e nc e Qu e n c h i n g o f Al b u mi n .

A s pe c t r of l uor i -
me t r i c e x p e r i me n t
MARI A TERESA MONTERO, JORDI HERNANDEZ
and JOAN ESTELRICH
Unitat de Fisicoqulmica
Departament de Farmgzcia
Universitat de Barcelona
Catalonia, Spain
Introduction
Molecules and at oms can absorb ultraviolet and visible electro-
magnetic radi at i on and be raised t o excited electronic states. The
reverse process, transition from an excited electronic state t o the
ground electronic with emission of radiation, is called lumin-
iscence. If the transition occurs bet ween two singlet states, the
emission is called fluorescence. Gr oups of at oms responsible for
fluorescence process are known as fluorophores.
Fluorimetric procedures which are simple and sensitive have
been devel oped for the quantitation of a wide variety of
substances of biological i mport ance. Nearl y all proteins exhibit
fluorescence in t he ultraviolet region. The potentially fluor-
escent ami no acid residues in prot ei ns are phenylalanine,
tyrosine and t rypt ophan. I n proteins containing all t hree amino
acids t he fluorescence spect rum is essentially t hat of t rypt ophan.
Even in human serum albumin which contains only one
t rypt ophan residue and ei ght een tyrosines, contributions by
tyrosines are relatively minor. 1
Background
A linear relationship exists bet ween fluorescence and concen-
tration in dilute solutions. However , at high concent rat i ons, this
proport i onal i t y no longer holds. Upon these conditions the so-
called ' i nner filter' effect has t o be considered. This effect occurs
at high concent rat i ons because the solution in the ' back' part of
t he sample cell does not receive the same intensity of excitation
energy as t he ' f r ont ' part of t he sample cell since t he intervening
solution has acted as an ' i nner filter' by absorbing most of the
exciting light. The solution as a whole is not uniformly excited
and there will be a reduct i on in luminescence emission. As a
general rule, it can be said t hat the total fluorescence intensity is
proport i onal t o concent rat i on when t he absorbance of the
sample is less t han about 0.05. On the ot her hand, if the
excitation and emission spectra of the sample overlap, the
luminescence emission can be absorbed by the sample itself. This
is the probl em of re-absorption.
Spect rofl uori met ry can also provi de qualitative i nformat i on
about prot ei n denat urat i on, and sensitive and precise infor-
mat i on about pr ot ei n- l i gand interactions. In t he latter case the
binding of small molecules t o proteins may be det ermi ned by
changes either in quant um yield or in fluorescence polarization.
Fluorescence quenchi ng refers t o any process which decreases
t he fluorescence intensity of a given substance. A variety of
processes can result in quenching. These included excited state
reactions, energy transfer, compl ex format i on, and collisional
quenching. 2 Nevertheless, the quenchi ng produced by the
binding of a molecule, known as quencher, t o t he fluorescent
molecule is a quenchi ng resulting f r om collisional encounters
bet ween t he fl uorophore and t he quencher, which is called
collisional or dynamic quenching. Static quenching, which is due
t o compl ex format i on, must also be considered. Fluorescence
quenchi ng has been widely studied bot h iis fundament al
phenomenon, and in the application of fluorescence t o bio-
chemical probl ems. These applications are due t o t he favorable
properties of t he process of quenching. Bot h static and dynamic
quenchi ng require mol ecul ar cont act bet ween the fl uorophore
and the quencher. I n t he case of collisional quenching, the
99
quencher must diffuse t o t he fl uorophore during t he lifetime of
t he excited state. Upon cont act , t he f l uor ophor e returns t o the
ground state, wi t hout emission of a phot on. I n t he case of static
quenchi ng a compl ex is f or med bet ween t he fl uorophore and the
quencher, and this compl ex is nonfl uorescent . I n either event,
the fl uorophore and quencher must be in contact. It is this
pri mary requi rement which results in t he numerous applications
of quenching. For example, quenchi ng measurement s can reveal
t he accesibility of fl uorophores t o quenchers. Furt hermore, it is
an i mport ant way t o investigate t he binding of drugs t o proteins
and their transport. The underst andi ng of pr ot ei n- l i gand bind-
ing is of prime interest in biochemistry and life sciences.
Experimental
The experi ment described here introduces the student t o some
basic principles of spect rofl uori met ry, as well as allowing t hem
t o visualize t he quenchi ng t hat caffeine produces on t he
fluorescence of human serum albumin.
Reagents
Caffeine was purchased from Merck ( W Ger many) , human
serum albumin ( HSA) from Sigma (St Louis, MO, USA) . All
experiments were carried out on a Perkin El mer 204 fluor-
escence spect rophot omet er. A mol ecul ar weight of 65 600 was
used for HSA preparat i ons, and the albumin concent rat i ons
were det ermi ned at 278 nm assuming a mol ar absorpt i on
coefficient of 33 700 M -1 cm -1 ( Ref 3).
Experimental Procedure
Step 1 Fl uorescence of serum albumin Firstly students must
det ermi ne t he excitation and emission wavelengths of albumin.
The albumin concent rat i on was 1 mg/ml. The observed ex-
citation wavelength was 295 nm and t he emission wavelength
was 335 nm. Students should underst and the i mport ance of slit
width. Slits are generally used t o minimize scattered light and the
emission slit must be as narrow as possible.
Step 2 Li neari t y bet ween fluorescence and concent rat i on Af t er
observing the fluorescent properties of albumin, the next step is
the det ermi nat i on of the upper limit of concent rat i on to t he
linear proport i onal i t y with fluorescence. Serial dilutions are
made from the initial prot ei n solution and t he relative fluor-
escence recorded. As shown in Table 1, if there is a proport i on-
ality bet ween bot h variables al t hough at high concent rat i ons
such proport i onal i t y no longer holds. With the values obt ai ned
from the more diluted solutions (samples 6- 10) , convent i onal
least-square analysis was carried out by regression of fluor-
escence against concent rat i on and a straight line with a
regression coefficient of 0.999 was obt ai ned. Fr om this straight
line the theoretical values for t he higher concent rat i ons may be
estimated. Nearly the same value experimentally and theor-
Table 1 Values obtained for fluorescence
Fluorescence
Sample ml Albumin ml Phosphate (%)*
1 3.00 0.00 102
2 2.50 0.50 94
3 2.00 1.00 78
4 1.50 1.50 58
5 1.00 2.00 40
6 0.75 2.25 29
7 0.50 2.50 20
8 0.25 2.75 9
9 0.10 2.90 4
10 0.00 3.00 0
(120)
(99)
(79)
(59)
*In brackets, theoretical values obtained from the straight line
obtained by linear regression of plotted points of samples 6 to 10
B I O C H E MI C A L E D U C A T I O N 18( 2) 1990
100
et i cal l y is obt ai ned for sampl es 3 and 4, wher eas t he exper -
i ment al resul t s ar e less t han t he pr edi ct ed ones for sampl es 1 and
2. It can be concl uded t hat at pr ot ei n concent r at i on bel ow
0.6 mg/ ml t her e exists a l i near pr opor t i onal i t y bet ween fl uor-
escence and concent r at i on.
Step 3 Abs ence of caffei ne f l uor escence Once t he fl uorescence
of al bumi n has been obser ved, t he same oper at i on is car r i ed out
wi t h caffei ne. Al t hough caffei ne shows an i nt ense absor pt i on in
t he ul t r avi ol et r egi on (274 nm) , it does not fl uoresce. At this
poi nt it is i nt er est i ng t o dr aw t he st udent s' at t ent i on t o t he fact
t hat not all abs or bed phot ons ar e emi t t ed as f l uor escence
because ot her pr ocesses can compet e wi t h f l uor escence and t he
exci t ed mol ecul e can l ose ener gy by ot her mechani sms. These
nonr adi at i ve deact i vat i on pr ocesses i ncl ude i nt er nal conver si on,
i nt er syst em crossi ng, f or mat i on of radi cal s as wel l as t r ansi t i ons
t o t he t r i pl et st at e. ~
Step 4 Fl uor escence quenchi ng of al bumi n The det er mi nat i on
of t he quenchi ng pr oduced by caffei ne on al bumi n is done by
mi xi ng a const ant amount of pr ot ei n wi t h var i abl e amount s of
caffei ne as shown in Tabl e 2. The fol l owi ng concent r at i ons ar e
sui t abl e: 1.5 x 10 -5 M for ser um al bumi n and 5.8 x 10 -3 M for
caffei ne. Thi r t y mi nut es af t er mi xi ng, t he f l uor escence in all
t ubes was det er mi ned. Fl uor escence of t he pr ot ei n sampl e t hat
cont ai ned onl y HS A in phos phat e buf f er was t aken as 100%
fl uorescence (F0), whi l e t he phos phat e buf f er gave t he 0%
f l uor escence.
r,
o
,'r
1 (X)
80
60
40
20
0 2
m
. m ~ m ~ m ~ w ~ e
I I I
4 6
[ C A F ] I ( ) - 4 M
/
I
I 0 12
Figure i Stern-Volmer plot obtained f rom the titration of 5 x
10 .6 M HSA with caffeine
i nt er act i ons occurri ng bet ween caffei ne and ser um al bumi n
when t he pr ot ei n is in its gr ound el ect r oni c st at e (st at i c
quenchi ng) and in its first exci t ed single st at e ( dynami c
quenchi ng) . 5 I n such ci r cumst ances it is mor e conveni ent to use
t he modi f i ed f or m of t he St e r n- Vol me r equat i on:
F d F = (1 + KD[CAF])(1 + Ks [ CAF] ) (2)
Table 2 Contents of tubes
Tube
Number ml Caffeine ml Al bumi n ml Phosphate
1 2.00 1.00 0.00
2 1.75 1.00 0.25
3 1.50 1.00 0.50
4 1.25 1.00 0.75
5 1.00 1.00 1.00
6 0.75 1.00 1.25
7 0. 70 1.00 1.30
8 0.60 1.00 1.40
9 0.50 1.00 1.50
10 0.45 1.00 1.55
11 0.40 1.00 1.60
12 0.35 1.00 1.65
13 0.30 1.00 1.70
14 0.25 1.00 1.75
15 0.20 1.00 1.80
16 0.15 1.00 1.85
17 0.10 1.00 1.90
18 0.05 1.00 1.95
19 0.00 1.00 2.00
Res ul t s and Di s cus s i on
I n or de r t o obt ai n t he dat a shown her e t he exper i ment was run in
t r i pl i cat e. Quenchi ng dat a wer e anal yzed usi ng t he St e r n -
Vol mer equat i on: 2
Fo/F = 1 + K[ CAF] (1)
wher e F0 has been def i ned above, F is t he f l uor escence of
pr ot ei n sol ut i on in pr esence of caffei ne at caffei ne concent r at i on
[ CAF] , and K is t he St e r n - Vo l me r const ant . Pl ot t i ng F0/F versus
[ CAF] t wo t ypes of pl ot s may be obt ai ned. A l i near St e r n -
Vol mer pl ot i ndi cat es t hat onl y one t ype of quenchi ng, dynami c,
occurs. Whe n t he same f l uor ophor e can be quenched both for
col l i si ons and by compl ex f or mat i on, an upwar d cur vat ur e is
obser ved. The pl ot obt ai ned wi t h ser um al bumi n is shown in Fi g.
1. I t devi at ed si gni fi cant l y f r om l i near i t y, whi ch is i ndi cat i ve of
wher e KD and Ks ar e t he dynami c and st at i c quenchi ng const ant s,
respect i vel y. This equat i on is second or der in quencher concen-
t r at i on, whi ch account s for t he upwar d cur vat ur e obser ved when
bot h st at i c and dynami c quenchi ng occur for t he same fl uoro-
phor e. Mul t i pl i cat i on of t he t er ms in par ent heses yi el ds
Fo/F = 1 + (K D + Ks)[CAF] + KDKs[CAF] 2 = 1 + Kapp[CAF]
(3)
wher e Kap p = (KD + Ks) + KDKs[ CAF] = (F0/F - 1/[CAF] is
t he appar ent quenchi ng const ant . A pl ot of Kapp versus [ CAF]
( Fi g 2) yi el ded a st rai ght line wi t h a specific sl ope. If a
f l uor escent mol ecul e t hat exper i ment s onl y one t ype of quench-
i ng had been used, such pl ot woul d have yi el ded a st rai ght line
par al l el t o x-axi s.
"7
~d
2 4 6 8 I 0
[ C A F ] 1 0 - 4 M
Figure 2 Variation of apparent quenching constant, Kapp, in
function of quencher concentration
Fr om t he r el at i ve fl uorescence val ues t he fract i on of t he
f l uor escence t hat has been quenched, Q, is obt ai ned as
O = (Fo - F)/100 (4)
Wi t h t hi s pa r a me t e r a new r epr es ent at i on can be made pl ot t i ng
Q versus [ CAF] / [ HSA] , wher e [ HSA] is t he concent r at i on of
B I O C H E M I C A L E D U C A T I O N 1 8 ( 2 ) 1 9 9 0
protein. Fig 3 shows this pl ot and f r om t hat t he limit quenchi ng
concent rat i on may be det ermi ned. A [CAF]/ [HSA] value of 320
was t he smallest relationship bet ween quencher and prot ei n t hat
pr oduced t he maximal quenchi ng and it is equivalent t o a
caffeine concent rat i on of 0.0016 M.
Q
wr pi m I-m
m"
0.8 d
/
0.6 !
~0.4
0.2
-=m " l .......
I O 0 I I I I
0 m 1 O0 2 300 400 500 600 700
[CAF]/[ASH]
Figure 3 Plot of fractional quenching, Q, in front of the
relationship between the concentrations of caffeine [CAF] and
albumin [HSA]
Event ual l y concent rat i ons of free (ie not bound t o albumin)
caffeine ([CAF]F) are calculated as
[CAF]F = [CAF] - Q[ HSA] (5)
where [CAF] is t he caffeine concent rat i on present in each tube,
[HSA] is t he albumin concent rat i on in t he samples (this value is
always 0.33 mg/ml), and Q is obt ai ned from eqn (4).
Fig 4 shows the dat a from the titration of albumin pl ot t ed as
t he reciprocal of fractional quenchi ng versus t he reciprocal of t he
free ligand concent rat i on. I n this plot, t he x intercept is - 1/ Kd,
K d being t he dissociation const ant of the prot ei n-quencher
complex. ~ We obt ai ned an average ~alue of 1.32 x 10 -3 M.
The experi ment can be compl et ed by determining t he number
of binding sites. For a system wi t hout interaction bet ween
binding sites one derives 7 an expression t o calculate the number
of binding sites (n):
Q/ [CAF]F = n/K d - Q/ K d (6)
A pl ot of Q/ [CAF]F against Q is known as a Scatchard plot
(Fig 5). I n this case t he pl ot is linear with an ordi nat e intercept of
n/Ka, an abscissa i nt ercept of n, and a slope of - 1/ Kd. This pl ot
provides a simple and conveni ent way of obt ai ni ng t he two
C'
2.30
2.05
1.80
1.55
1.30
1.05
0.80
-2
a /
. Y
s I i / I I /
m~ ' m "m
0 2 4 6 8 10
I/[CAF] F x 10~M
Figure 4 Plot of the reciprocal of fractional quenching, Q, versus
the reciprocal of the concentration of non bound caffeine [CAF]r
BI OCHEMI CAL EDUCATI ON 18( 2) 1990
8OOO
7000
a. 6000
~ 5 ~
r~
--'~ 4000
~ 3000
2000
1 0 0 0 ~ 1 ~
1 , , , , , " - , ,
101
0 0.2 0.4 0.6 0.8 1.0
Q
1.2
Figure 5 Scatchard plot f or identical, independent binding sites of
caffeine to human serum albumin
paramet ers t hat characterize t he binding equilibria. The esti-
mat ed value of n is 1.1 and thus albumin has one binding site to
caffeine.
In summary, this experi ment offers a simple and relatively
inexpensive procedure for t he study and underst andi ng of the
basic principles of prot ei n-fl uorescence quenchi ng which is
manageabl e in the typical 3- or 4-h l aborat ory period.
References
1Steiner, R F and Edelhoch, H (1963) Biochim Biophys Acta 66,
341-355
2Lakowicz, J R (1986) in 'Principles of Fluorescence Spectroscopy',
Plenum Press, New York and London, Chapter 9
3Kirschenbaum, D M (1977) Anal Biochem 81,220-246
4Lott, P F (1974) J Chem Educ 51, A315-A364
5Gheorghiou, S (1981) in ' Modem Fluorescence Spectroscopy' (Wehry,
E L, Editor), Plenum Press, New York and London, Vol 3, Chapter 6
6Royer, R E, Kibirige, M, Tafoya, C R, Deck, L M and Vander Jagt,
D L (1988) J Pharm Sci 77, 237-240
7Cantor, C R and Schimmel, P R (1980) in 'Biophysical Chemistry' W H
Freeman and Co, San Francisco, Part III, Chapter 15
Enzyme Kinetics - - The Steady-State Observed
P AUL D BUCKL E Y, L E ONAR D F B L AC KWE L L ,
MI CHAEL F DUNNf and JEREMY P HILL
Depart ment o f Chemistry and Bi ochemi st ry
Massey University
Pal merst on Nort h, Ne w Zeal and
and
t Bi ochemi st ry Depart ment
UCR, Riverside, CA 92521, USA
Introduction
The vast field of steady-state kinetics has been founded on t he
assumption t hat the concent rat i ons of intermediates on t he
pat hway of an enzyme catalysed react i on rapidly build up and
t hen remain const ant until depl et i on of a substrate occurs.
Mathematically, we write for each i nt ermedi at e on t he pat hway:
d[Int ermedi at e] = 0 (1)
dt
I n this way t he set of differential equat i ons which describe the
change in concent rat i on of t he intermediates with time, immedi-