370 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 35984

BINDING OF LIGANDS BY PROTEINS: A SIMPLE METHOD W I T H

S E P H A D E X GEL

MASAAKI H I R O S E AND Y A S U Y U K I KANO

Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto (Japan)

(Received Ju ly 2nd, 1971)

SUMMARY

A simple procedure for the measurement of ligand binding by proteins was devised with Sephadex gel; this procedure differs from the more common method of gel filtration.

INTRODUCTION

The most commonly employed procedure for the measurement of ligand binding by proteins is dialysis equilibrium. This is based on the fact that the mem- brane is permeable to ligands, but not to proteins. A system similar to dialysis equi- librium can be accomplished with the appropriate Sephadex gel, since ligands are diffusible inside the gel and proteins are not. The equilibrium of ligands between the inside and the outside of the gel is attained rapidly. This is the advantage for binding experiments.

The procedure to be described in this report differs from the method of HUMMEL AND DRYER 1 in that the gel filtration is not carried out. The solution containing the protein and/or the ligand was added to the fixed weight of the dried gel. The binding of the ligand by the protein was determined from the amount of the protein and the ligand outside the gel.

With this procedure the binding of methyl orange by bovine serum albumin was examined in order to compare the result with data obtained from the dialysis equilibrium by KLOTZ et al. 2.

MATERIALS AND METHODS

Crystalline bovine serum albumin was purchased from Sigma, and methyl orange from Merck. Sephadex G-5o (coarse) was washed with water, and the water in the gel was exchanged with ethanol. After the gel was dried at room temperature, ethanol was removed by heating in an oven at 80 ° for 3 h.

Biochim. Biophys. Acla, 251 (i97i) 376-379

A S I M P L E M E T H O D F O R B I N D I N G S T U D I E S 377

To o.I g of the dried gel, 0.75 ml of o.I M phosphate buffer (pH 5.7) was added. After swelling of the gel was completed by standing for about 4 h or more at room temperature (the t ime for swelling of the gel was tested by measuring the concentra- tion of the protein outside the gel at several t ime intervals), 0.45 ml of the buffer solution containing the protein and/or methyl orange was added to the gel solution. The solution was incubated for IO min at 25 ° with continuous stirring (it was con- firmed by measuring the concentration of the ligand outside the gel that a Io-min incubation was enough to equilibrate the ligand concentration between the outside and the inside of the gel). o.15-o.2o ml of the solution outside the gel was withdrawn with a pipette and the solution was diluted with 2.0-2.5 ml of the buffer. The concen- trations of the protein and methyl orange were determined from the absorbances at 280 nm and 460 nm, respectively (bovine serum albumin, e (280 n m ) = 4.36" lO 7 cm2/ mole; methyl orange, e (460 nm) = 2.58.1o 7 cm2/mole).

R E S U L T S A N D D I S C U S S I O N

The outer volume of the gel, Vo, is estimated from the following equation.

V Vo = - - (i)

(z

Where V is the total volume of the solution added to the dried gel, and a is the ratio of the concentration of the protein outside the gel (P/Vo; P, the total amount of the protein) to that of the protein solution added to the dried gel (P/V). Therefore, the apparent inner volume of the gel, Vi, is as follows.

I '

When L, Lo* and Li are defined as the amount of the ligand added and the amounts outside the gel and inside the gel, respectively, the following equations are obtained.

/SLVo f lL L o = (3)

V a

/~ is the ratio of the concentration of the ligand outside the gel (Lo/Vo) to that of the solution of the ligand added (L/V). When the weight of the dried gel and the volume of the solution added to the gel are kept constant, a must be constant, but fl should be varied by the binding of the ligand by the protein. I f fi' is defined as fi in the ab- sence of the protein, y in the following equation must be constant.

L i a - - f l ' (5)

Lo •'

In this case, Lo is the amount of the free ligand outside the gel. From the Eqns. 3-5, Lo(free) is given as follows, since the ligand inside the gel should exist as free-form.

= I L / 5 ' ( a - - fl) L o ( f r e e ) - L i = (6) ~, a(a - ~') * L o - - L o ( f ree) + L o ( b o u n d ) .

Biochim. Biophys. dcta, 251 (1971) 3 7 6 - 3 7 9

378 M. HIROSE, Y. KANO

The amount of the ligand bound by the protein, Lo(bound), is as follows.

Lo (bound) = Lo -- Lo (free) = LI5 -- fl' _/~, (7)

The concentrat ion of the free-ligand outside the gel, [Lo(free)J, is given as follows.

Lo(free) f l ' (a - - fl) [Lo (free)] (8)

Vo a - fl'

For the determination of the dissociation constant, K, between the ligand and the protein and N, the max imum number of ligand molecules bound per protein molecule, the following equation 2 was used.

I K I I

+ - - (9) R N [Lo(free)] N

R is defined as Lo(bound)/P. Fig. 1 shows the plot of the concentrat ion of the protein outside the gel (P/Vo)

v s . tha t of the protein added (P/V). The linearity of the plot suggested that the volume outside the gel was not varied by the addit ion of the protein. The value of a , 2.Ol, was obtained from the slope of the line. This indicated tha t Vo was o.597 ml (V = 1.2o ml).

8O

6C

40

.G E _~ 20

8 o~ 2'o 4b

Cohen. of albumin added (pM)

E o~

o]

o 0.4 g c o o

o.2 E

d

o ~o~

3

2

1

o:2 o:4 ' o'.6 Concn. of methyl orange added (mM)

Fig. I. The l inear re la t ion be tween the concen t r a t ion of a l bumi n added (P/V) and t h a t of a l bumi n ou ts ide the gel (P/Vo). The weak absorbance of the solut ion ob ta ined from the gel solut ion in the absence of the p ro te in was s u b t r a c t e d from the va lues ob ta ined in the presence of the protein.

Fig. 2. The p lo t of the concen t ra t ion of m e t h y l orange outs ide the gel (Lo/Vo) vs. t h a t of m e t h y l orange added (L/V). The w e a k absorbance of the solut ion ob ta ined from the gel solut ion in the absence of m e t h y l orange (in the presence of a l bum in in the l ines 2 and 3) was sub t r ac t ed f rom the va lues ob ta ined in the presence of m e t h y l orange. Addi t ion : l ine I, none; line 2, 12 nmoles of a lbumin ; l ine 3, 24 nmoles of a lbumin.

B i o c h i m . B i o p h y s . A c t a , 251 (1971) 376-379

A SIMPLE METHOD FOR BINDING STUDIES 379

Fig. 2 shows t h a t the plot of the concent ra t ion of me thy l orange outs ide the gel (Lo/Vo) vs. t h a t of m e t h y l orange added (L/V) was l inear in the absence of the protein. This i nd i ca t ed t h a t 7 in Eqn. 5 was cons tan t wi th in the concent ra t ion range of me thy l orange used. F r o m the slope of l ine i , fl' ~ 0.622 was obta ined . Fig. 2 also shows t h a t the concen t ra t ion of me thy l orange outs ide the gel was increased b y the add i t ion of the prote in . The values of f l in lines 2 and 3 were ob ta ined from the ra t io of Lo/Vo to L / V .

0.6

0.4

0,2

t / o ~ 2b 3b

Fig. 3. The plot of IIR vs. l/[Lo (flee)]. The values of [Lo (free)] were calculated from Eqn. 8 in the text. The values of R were obtained from the ratio of Lo (bound) (see Eqn. 7) to the amount of albumin (i2 nmoles or 24 nmoles). From the intercept of the line on the ordinate and the abscissa, N = 20.0 and K = o.4o mM were obtained, respectively (see Eqn. 9)- O, 12 nmoles of albumin; O, 24 nmoles of albumin.

Fig. 3 shows t h a t N is 2o and K is o.4o mM. These values agreed well wi th the values ob ta ined f rom dia lys is equi l ibr ium b y KLOTZ et al. 2 ( N = 22.4, K = 0.448 mM). This indica tes t ha t the me thod descr ibed here is appl icab le to s tudies of the b ind ing of l igands b y macromolecules .

The d i s advan t ages of th is me thod are t h a t the concen t ra t ion of l igands mus t be measu red in the presence of p ro te ins and a high concen t ra t ion of e lec t ro ly te mus t be present to min imize the Donnan effect. The ma jo r a d v a n t a g e of this m e t h o d is t h a t b ind ing of l igands b y pro te ins can be r ap id ly measured wi th a s imple t r e a t m e n t and wi thou t any pa r t i cu la r appa ra tus . Using this method , b ind ing s tudies for a wide range of molecular weights of l igands and macromolecules will be made possible b y selection of a su i tab le gel.

ACKNOWLEDGMENT

We wish to t h a n k Dr. E. Sugimoto and Professor H. Chiba for helpful sugges- t ions and discussions.

REFERENCES

I J. P. HUMMEL AND W. J. DREYER, Biochi~q~. Biophys. Acta, 63 (1962) 53 o. 2 I. M. KLOTZ, F. M. WALKER AND R. B. PIVAN, J. Am. Chem. Soc., 68 (1946) 1486.

Biochim. Biophys. Acta, 251 (1971) 376-379