Fluorescence Quenching of Albumin. A spectrofluori- metric experiment

MARIA TERESA MONTERO, JORDI HERNANDEZ and JOAN ESTELRICH

Unitat de Fisicoqulmica Departament de Farmgzcia Universitat de Barcelona Catalonia, Spain

Introduction Molecules and atoms can absorb ultraviolet and visible electro- magnetic radiation and be raised to excited electronic states. The reverse process, transition from an excited electronic state to the ground electronic with emission of radiation, is called lumin- iscence. If the transition occurs between two singlet states, the emission is called fluorescence. Groups of atoms responsible for fluorescence process are known as fluorophores.

Fluorimetric procedures which are simple and sensitive have been developed for the quantitation of a wide variety of substances of biological importance. Nearly all proteins exhibit fluorescence in the ultraviolet region. The potentially fluor- escent amino acid residues in proteins are phenylalanine, tyrosine and tryptophan. In proteins containing all three amino acids the fluorescence spectrum is essentially that of tryptophan. Even in human serum albumin which contains only one tryptophan residue and eighteen tyrosines, contributions by tyrosines are relatively minor. 1

Background A linear relationship exists between fluorescence and concen- tration in dilute solutions. However, at high concentrations, this proportionality no longer holds. Upon these conditions the so- called 'inner filter' effect has to be considered. This effect occurs at high concentrations because the solution in the 'back' part of the sample cell does not receive the same intensity of excitation energy as the 'front' part of the sample cell since the intervening solution has acted as an 'inner filter' by absorbing most of the exciting light. The solution as a whole is not uniformly excited and there will be a reduction in luminescence emission. As a general rule, it can be said that the total fluorescence intensity is proportional to concentration when the absorbance of the sample is less than about 0.05. On the other hand, if the excitation and emission spectra of the sample overlap, the luminescence emission can be absorbed by the sample itself. This is the problem of re-absorption.

Spectrofluorimetry can also provide qualitative information about protein denaturation, and sensitive and precise infor- mation about protein-ligand interactions. In the latter case the binding of small molecules to proteins may be determined by changes either in quantum yield or in fluorescence polarization.

Fluorescence quenching refers to any process which decreases the fluorescence intensity of a given substance. A variety of processes can result in quenching. These included excited state reactions, energy transfer, complex formation, and collisional quenching. 2 Nevertheless, the quenching produced by the binding of a molecule, known as quencher, to the fluorescent molecule is a quenching resulting from collisional encounters between the fluorophore and the quencher, which is called collisional or dynamic quenching. Static quenching, which is due to complex formation, must also be considered. Fluorescence quenching has been widely studied both iis fundamental phenomenon, and in the application of fluorescence to bio- chemical problems. These applications are due to the favorable properties of the process of quenching. Both static and dynamic quenching require molecular contact between the fluorophore and the quencher. In the case of collisional quenching, the

99

quencher must diffuse to the fluorophore during the lifetime of the excited state. Upon contact, the fluorophore returns to the ground state, without emission of a photon. In the case of static quenching a complex is formed between the fluorophore and the quencher, and this complex is nonfluorescent. In either event, the fluorophore and quencher must be in contact. It is this primary requirement which results in the numerous applications of quenching. For example, quenching measurements can reveal the accesibility of fluorophores to quenchers. Furthermore, it is an important way to investigate the binding of drugs to proteins and their transport. The understanding of protein-ligand bind- ing is of prime interest in biochemistry and life sciences.

Experimental The experiment described here introduces the student to some basic principles of spectrofluorimetry, as well as allowing them to visualize the quenching that caffeine produces on the fluorescence of human serum albumin.

Reagents Caffeine was purchased from Merck (W Germany), human serum albumin (HSA) from Sigma (St Louis, MO, USA). All experiments were carried out on a Perkin Elmer 204 fluor- escence spectrophotometer. A molecular weight of 65 600 was used for HSA preparations, and the albumin concentrations were determined at 278 nm assuming a molar absorption coefficient of 33 700 M -1 cm -1 (Ref 3).

Experimental Procedure Step 1 Fluorescence of serum albumin Firstly students must determine the excitation and emission wavelengths of albumin. The albumin concentration was 1 mg/ml. The observed ex- citation wavelength was 295 nm and the emission wavelength was 335 nm. Students should understand the importance of slit width. Slits are generally used to minimize scattered light and the emission slit must be as narrow as possible. Step 2 Linearity between fluorescence and concentration After observing the fluorescent properties of albumin, the next step is the determination of the upper limit of concentration to the linear proportionality with fluorescence. Serial dilutions are made from the initial protein solution and the relative fluor- escence recorded. As shown in Table 1, if there is a proportion- ality between both variables although at high concentrations such proportionality no longer holds. With the values obtained from the more diluted solutions (samples 6-10), conventional least-square analysis was carried out by regression of fluor- escence against concentration and a straight line with a regression coefficient of 0.999 was obtained. From this straight line the theoretical values for the higher concentrations 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

BIOCHEMICAL EDUCATION 18(2) 1990

100

etically is obtained for samples 3 and 4, whereas the exper- imental results are less than the predicted ones for samples 1 and 2. It can be concluded that at protein concentration below 0.6 mg/ml there exists a linear proportionality between fluor- escence and concentration. Step 3 Absence of caffeine fluorescence Once the fluorescence of albumin has been observed, the same operation is carried out with caffeine. Although caffeine shows an intense absorption in the ultraviolet region (274 nm), it does not fluoresce. At this point it is interesting to draw the students' attention to the fact that not all absorbed photons are emitted as fluorescence because other processes can compete with fluorescence and the excited molecule can lose energy by other mechanisms. These nonradiative deactivation processes include internal conversion, intersystem crossing, formation of radicals as well as transitions to the triplet state. ~ Step 4 Fluorescence quenching of albumin The determination of the quenching produced by caffeine on albumin is done by mixing a constant amount of protein with variable amounts of caffeine as shown in Table 2. The following concentrations are suitable: 1.5 x 10 -5 M for serum albumin and 5.8 x 10 -3 M for caffeine. Thirty minutes after mixing, the fluorescence in all tubes was determined. Fluorescence of the protein sample that contained only HSA in phosphate buffer was taken as 100% fluorescence (F0), while the phosphate buffer gave the 0% fluorescence.

r,

o ,'r

1 (X)

80

60

40

20

0 2

m

.m~m~m ~w~e

I I I 4 6

[CAF] I() -4 M

/ I

I0 12

Figure i Stern-Volmer plot obtained from the titration of 5 x 10 .6 M HSA with caffeine

interactions occurring between caffeine and serum albumin when the protein is in its ground electronic state (static quenching) and in its first excited single state (dynamic quenching). 5 In such circumstances it is more convenient to use the modified form of the Stern-Volmer equation:

FdF = (1 + KD[CAF])(1 + Ks[CAF]) (2)

Table 2 Contents of tubes

Tube Number ml Caffeine ml Albumin 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

Results and Discussion In order to obtain the data shown here the experiment was run in triplicate. Quenching data were analyzed using the Stern- Volmer equation: 2

Fo/F = 1 + K[CAF] (1)

where F0 has been defined above, F is the fluorescence of protein solution in presence of caffeine at caffeine concentration [CAF], and K is the Stern-Volmer constant. Plotting F0/F versus [CAF] two types of plots may be obtained. A linear Stern- Volmer plot indicates that only one type of quenching, dynamic, occurs. When the same fluorophore can be quenched both for collisions and by complex formation, an upward curvature is observed. The plot obtained with serum albumin is shown in Fig. 1. It deviated significantly from linearity, which is indicative of

where KD and Ks are the dynamic and static quenching constants, respectively. This equation is second order in quencher concen- tration, which accounts for the upward curvature observed when both static and dynamic quenching occur for the same fluoro- phore. Multiplication of the terms in parentheses yields

Fo/F = 1 + (K D + Ks)[CAF] + KDKs[CAF] 2 = 1 + Kapp[CAF] (3)

where Kap p = (KD + Ks) + KDKs[CAF] = (F0/F - 1/[CAF] is the apparent quenching constant. A plot of Kapp versus [CAF] (Fig 2) yielded a straight line with a specific slope. If a fluorescent molecule that experiments only one type of quench- ing had been used, such plot would have yielded a straight line parallel to x-axis.

"7

~d

2 4 6 8 I0

[CAF] 10 -4 M

Figure 2 Variation of apparent quenching constant, Kapp, in function of quencher concentration

From the relative fluorescence values the fraction of the fluorescence that has been quenched, Q, is obtained as

O = (Fo - F)/100 (4)

With this parameter a new representation can be made plotting Q versus [CAF]/[HSA], where [HSA] is the concentration of

BIOCHEMICAL EDUCATION 18(2) 1990

protein. Fig 3 shows this plot and from that the limit quenching concentration may be determined. A [CAF]/[HSA] value of 320 was the smallest relationship between quencher and protein that produced the maximal quenching and it is equivalent to a caffeine concentration of 0.0016 M.

Q

wrpim I-m m"

0.8 d /

0.6 !

~0.4

0.2

-=m "l .......

I O0 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]

Eventually concentrations of free (ie not bound to albumin) caffeine ([CAF]F) are calculated as

[CAF]F = [CAF] - Q[HSA] (5)

where [CAF] is the caffeine concentration present in each tube, [HSA] is the albumin concentration in the samples (this value is always 0.33 mg/ml), and Q is obtained from eqn (4).

Fig 4 shows the data from the titration of albumin plotted as the reciprocal of fractional quenching versus the reciprocal of the free ligand concentration. In this plot, the x intercept is -1/Kd, K d being the dissociation constant of the protein-quencher complex. ~ We obtained an average ~alue of 1.32 x 10 -3 M.

The experiment can be completed by determining the number of binding sites. For a system without interaction between binding sites one derives 7 an expression to calculate the number of binding sites (n):

Q/[CAF]F = n/K d - Q/K d (6)

A plot of Q/[CAF]F against Q is known as a Scatchard plot (Fig 5). In this case the plot is linear with an ordinate intercept of n/Ka, an abscissa intercept of n, and a slope of -1/Kd. This plot provides a simple and convenient way of obtaining the 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

BIOCHEMICAL EDUCATION 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 for identical, independent binding sites of caffeine to human serum albumin

parameters that characterize the binding equilibria. The esti- mated value of n is 1.1 and thus albumin has one binding site to caffeine.

In summary, this experiment offers a simple and relatively inexpensive procedure for the study and understanding of the basic principles of protein-fluorescence quenching which is manageable in the typical 3- or 4-h laboratory 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

PAUL D BUCKLEY, LEONARD F BLACKWELL , MICHAEL F DUNNf and JEREMY P HILL

Department of Chemistry and Biochemistry Massey University Palmerston North, New Zealand and t Biochemistry Department UCR, Riverside, CA 92521, USA

Introduction The vast field of steady-state kinetics has been founded on the assumption that the concentrations of intermediates on the pathway of an enzyme catalysed reaction rapidly build up and then remain constant until depletion of a substrate occurs. Mathematically, we write for each intermediate on the pathway:

d[Intermediate] = 0 (1) dt

In this way the set of differential equations which describe the change in concentration of the intermediates with time, immedi-