- Email: [email protected]
Determination of binding constants by high-performance gel filtration
Journal of Chromatography, 404 (1987) 254-257 Elsevier Science Publishers B.V., Amsterdam CHROM.
Printed
in The Netherlands
19 702
Note
Determination
of binding
constants
by high-performance
gel filtration
Yu. A. MOTORIN A. N. Bach Institute of Biochemistry, Academy of Sciences (U.S.S.R.) (Received
USSR,
Leninsky pr. 33, Moscow 117071
May 6th. 1987)
Gel filtration is one of the most commonly used non-kinetic methods for the determination of association constants of ligands with proteins and nucleic acids1-4. The most widely used modification of this method is based on the determination of the trough area of the ligand which appears after the protein peak on a column equilibrated with ligand. This allows an estimation of both the association constant and the stoichiometry of labile complexes. The main disadvantages of this method are that it is laborious and large amounts of enzyme and ligand are required, which limits its application to readily available reagents5s6. The aim of this work is to demonstrate a modification of the constant determination method by gel filtration, which allows a significant decrease in the amount of protein and time required due to the complete automation of the experiment. EXPERIMENTAL
Sephadex G-25 Superfine (Pharmacia), morpholinoethanesulphonic acid (MES), pig skeletal muscle lactate dehydrogenase (LDH) and nicotinamide-adenine dinucleotide (NAD) (Reanal, Hungary) were employed. The other reagents were analytical grade. The buffers were: A, 25 mM MES-sodium hydroxide, pH 6.0, + 1 mM magnesium chloride; B, 77 pM NAD in buffer A. A fast protein liquid chromatography (FPLC) system (Pharmacia) comprising two P-500 and P-l pumps, controller LCC-500, a motor valve MV-7, and UV-1 monitor and a column HR IO/l0 (100 mm x 10 mm I.D.) packed with Sephadex G-25 SF7 was used for determination of the association constants. A personal computer Apple-2E, connected to the controller LCC-500 of the FPLC system with a standard interface RS-232C, was used for process control and evaluation of the experimental data. The programs were written in BASIC, using interfacing software (Pharmacia) for the LCC-500 and Apple-2E computer. The experiments were performed as follows. The column was equilibrated with buffer containing a definite ligand concentration, and an enzyme aliquot (100 ~1, 6.7 mg/ml in buffer A) was automatically applied. At the same time, recording and integration of the trough area were started by the Apple-2E computer. The elution rate was 2 ml/min. After the elution was complete, the computer defined a new ligand concentration and the process was repeated. Simultaneously, the experimental data 0021-96731871SO3.50
0
1987 Elsevier Science Publishers
B.V.
NOTES
255
0
10 VOLUMt. ml
Fig. 1. Elution profile of LDH on a Sephadex G-25 Superfine column HR lo/l0 equilibrated with 30 PM NAD solution. (A) 100 pl LDH solution (650 pg); (B) 100 ~1 buffer. Buffer: 25 mM MESsodium hydroxide, pH 6.0, 1 mM magnesium chloride.
and results of the integration were stored on a diskette. Correction of the trough area for the baseline drift was made, if necessary. Corrections for the dilution effect were also made, equal volumes of buffer A being applied’to .the column instead of the , enzyme solution.
Fig. 2. Effect of the concentration of NAD on the trough area. Curves: I, in the presence calibration curve for the effect of dilution: 3. curve I corrected for the dilution.
LDH;
2,
256
NOTES
The association constants and stoichiometry method of non-linear regression*.
of binding were calculated by the
RESULTS
A typical chromatogram of lactate dehydrogenase on the Sephadex G-25 column equilibrated with NAD is presented on Fig. 1. The area of the reversed peak (trough) of NAD which follows the enzyme peak is significantly more than that of the dilution peak. The trough is well separated from the protein peak by a horizontal plateau at the level of the baseline. Fig. 2 demonstrates the relationship between the trough area and ligand concentration. The effect of dilution is practically linearly related to the ligand concentration over the range selected (curve 2), whereas in the presence of enzyme the dependence is hyperbolic (curve 1). The association constant and stoichiometry of NAD binding to LDH were calculated from the data corrected for the effect of dilution. The association constant was found to be 7.7 f 2.1 PM, and the stoichiometry was one molecule of NAD per five molecules of monomeric enzyme, or, approximately, one molecule per tetramer. DISCUSSION
As mentioned above, the use of gel filtration for constant determination is laborious and requires significant amounts of both macromolecular components and ligands. In this modification, the use of the small-volume high-resolution column decreases significantly the amount of enzyme required (about 3 mg of enzyme for ten experimental points). The amount of ligand required is also reduced. Due to the small volume and high resolution of the column and high flow-rates, the analysis time is decreased from hours to about 10 min. Use of the Apple-2E computer coupled to the FPLC system results in a fully automatic process and avoids mistakes caused by incomplete equilibration of the column, and, differences in the volumes of enzyme solution injected. Another important property is that no pre-equilibration of enzyme with ligand is required. This excludes inaccuracies connected with differences in ligand concentration in the enzyme solution and column buffer, but limits the use of this method to cases where the equilibrium between free and bound ligand is rapidly achieved. The value for the binding constant of NAD to LDH corresponds well to those obtained by other methodsg. The low stoichiometry of binding may be due to the presence of non-active protein molecules in the enzyme preparation. This method allows the determination constant having values between 1 and 100 PM. The lower limit is determined by the sensitivity of optical detection unit, the upper one by the effect of dilution, which becomes very significant. The use of more sensitive (fluorescence or radioisotopic) methods of detection may extend the application of this method. ACKNOWLEDGEMENT
I thank Dr. K. L. Gladilin for his interest in this work.
NOTES REFERENCES 1 2 3 4 5 6 I 8 9
G. C. Wood and P. F. Cooper, Chromatogr. Rev., 12 (1970) 88. J. D. Hummel and W. J. Dreyer, Biochim. Biophys. Acta, 63 (1962) 530. J. Clausen, J. Pharm. Exp. Ther., 153 (1966) 167. P. F. Cooper and G. C. Wood, J. Pharm. Pharmacol., 20 (1968) 1508. C. F. Fairclough and J. S. Fruton, Biochemistry, 5 (1966) 673. W. Hoffmann and U. Westphal, Anal. B&hem., 32 (1969) 48. R. Penney, High Pecformance Desalting BLT News, 18 (1985) 3p. R. G. Duggleby, Anal. Biochem., 110 (1981) 9. J. Everse and N. 0. Kaplan, Adv. Enzymol., 37 (1973) 61.
251
Printed
in The Netherlands
19 702
Note
Determination
of binding
constants
by high-performance
gel filtration
Yu. A. MOTORIN A. N. Bach Institute of Biochemistry, Academy of Sciences (U.S.S.R.) (Received
USSR,
Leninsky pr. 33, Moscow 117071
May 6th. 1987)
Gel filtration is one of the most commonly used non-kinetic methods for the determination of association constants of ligands with proteins and nucleic acids1-4. The most widely used modification of this method is based on the determination of the trough area of the ligand which appears after the protein peak on a column equilibrated with ligand. This allows an estimation of both the association constant and the stoichiometry of labile complexes. The main disadvantages of this method are that it is laborious and large amounts of enzyme and ligand are required, which limits its application to readily available reagents5s6. The aim of this work is to demonstrate a modification of the constant determination method by gel filtration, which allows a significant decrease in the amount of protein and time required due to the complete automation of the experiment. EXPERIMENTAL
Sephadex G-25 Superfine (Pharmacia), morpholinoethanesulphonic acid (MES), pig skeletal muscle lactate dehydrogenase (LDH) and nicotinamide-adenine dinucleotide (NAD) (Reanal, Hungary) were employed. The other reagents were analytical grade. The buffers were: A, 25 mM MES-sodium hydroxide, pH 6.0, + 1 mM magnesium chloride; B, 77 pM NAD in buffer A. A fast protein liquid chromatography (FPLC) system (Pharmacia) comprising two P-500 and P-l pumps, controller LCC-500, a motor valve MV-7, and UV-1 monitor and a column HR IO/l0 (100 mm x 10 mm I.D.) packed with Sephadex G-25 SF7 was used for determination of the association constants. A personal computer Apple-2E, connected to the controller LCC-500 of the FPLC system with a standard interface RS-232C, was used for process control and evaluation of the experimental data. The programs were written in BASIC, using interfacing software (Pharmacia) for the LCC-500 and Apple-2E computer. The experiments were performed as follows. The column was equilibrated with buffer containing a definite ligand concentration, and an enzyme aliquot (100 ~1, 6.7 mg/ml in buffer A) was automatically applied. At the same time, recording and integration of the trough area were started by the Apple-2E computer. The elution rate was 2 ml/min. After the elution was complete, the computer defined a new ligand concentration and the process was repeated. Simultaneously, the experimental data 0021-96731871SO3.50
0
1987 Elsevier Science Publishers
B.V.
NOTES
255
0
10 VOLUMt. ml
Fig. 1. Elution profile of LDH on a Sephadex G-25 Superfine column HR lo/l0 equilibrated with 30 PM NAD solution. (A) 100 pl LDH solution (650 pg); (B) 100 ~1 buffer. Buffer: 25 mM MESsodium hydroxide, pH 6.0, 1 mM magnesium chloride.
and results of the integration were stored on a diskette. Correction of the trough area for the baseline drift was made, if necessary. Corrections for the dilution effect were also made, equal volumes of buffer A being applied’to .the column instead of the , enzyme solution.
Fig. 2. Effect of the concentration of NAD on the trough area. Curves: I, in the presence calibration curve for the effect of dilution: 3. curve I corrected for the dilution.
LDH;
2,
256
NOTES
The association constants and stoichiometry method of non-linear regression*.
of binding were calculated by the
RESULTS
A typical chromatogram of lactate dehydrogenase on the Sephadex G-25 column equilibrated with NAD is presented on Fig. 1. The area of the reversed peak (trough) of NAD which follows the enzyme peak is significantly more than that of the dilution peak. The trough is well separated from the protein peak by a horizontal plateau at the level of the baseline. Fig. 2 demonstrates the relationship between the trough area and ligand concentration. The effect of dilution is practically linearly related to the ligand concentration over the range selected (curve 2), whereas in the presence of enzyme the dependence is hyperbolic (curve 1). The association constant and stoichiometry of NAD binding to LDH were calculated from the data corrected for the effect of dilution. The association constant was found to be 7.7 f 2.1 PM, and the stoichiometry was one molecule of NAD per five molecules of monomeric enzyme, or, approximately, one molecule per tetramer. DISCUSSION
As mentioned above, the use of gel filtration for constant determination is laborious and requires significant amounts of both macromolecular components and ligands. In this modification, the use of the small-volume high-resolution column decreases significantly the amount of enzyme required (about 3 mg of enzyme for ten experimental points). The amount of ligand required is also reduced. Due to the small volume and high resolution of the column and high flow-rates, the analysis time is decreased from hours to about 10 min. Use of the Apple-2E computer coupled to the FPLC system results in a fully automatic process and avoids mistakes caused by incomplete equilibration of the column, and, differences in the volumes of enzyme solution injected. Another important property is that no pre-equilibration of enzyme with ligand is required. This excludes inaccuracies connected with differences in ligand concentration in the enzyme solution and column buffer, but limits the use of this method to cases where the equilibrium between free and bound ligand is rapidly achieved. The value for the binding constant of NAD to LDH corresponds well to those obtained by other methodsg. The low stoichiometry of binding may be due to the presence of non-active protein molecules in the enzyme preparation. This method allows the determination constant having values between 1 and 100 PM. The lower limit is determined by the sensitivity of optical detection unit, the upper one by the effect of dilution, which becomes very significant. The use of more sensitive (fluorescence or radioisotopic) methods of detection may extend the application of this method. ACKNOWLEDGEMENT
I thank Dr. K. L. Gladilin for his interest in this work.
NOTES REFERENCES 1 2 3 4 5 6 I 8 9
G. C. Wood and P. F. Cooper, Chromatogr. Rev., 12 (1970) 88. J. D. Hummel and W. J. Dreyer, Biochim. Biophys. Acta, 63 (1962) 530. J. Clausen, J. Pharm. Exp. Ther., 153 (1966) 167. P. F. Cooper and G. C. Wood, J. Pharm. Pharmacol., 20 (1968) 1508. C. F. Fairclough and J. S. Fruton, Biochemistry, 5 (1966) 673. W. Hoffmann and U. Westphal, Anal. B&hem., 32 (1969) 48. R. Penney, High Pecformance Desalting BLT News, 18 (1985) 3p. R. G. Duggleby, Anal. Biochem., 110 (1981) 9. J. Everse and N. 0. Kaplan, Adv. Enzymol., 37 (1973) 61.
251