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Complex formation between chlorpromazine and adenosine triphosphate
ARCHIVES
OF
BIOCHEMISTRY
Complex
AND
Formation
BIOPHYSICS
321-324 (1965)
109,
between
Chlorpromazine
and
Adenosine
Triphosphate IRA BLEI Research Division,
Melpar,
Received
Inc., Falls
September
Church,
Virginia
2, 1964
The surface tensions of solutions of chlorpromazine have been shown to decrease in the presence of ATP. The data indicate reversible formation of a complex of greater surface activity than chlorpromazine alone. The extent of surface chemical complex formation of chlorpromazine with homologs of ATP of shorter phosphate chain length indicated that the reaction with ATP was specific and probably was a result of salt formation between the phosphate groups of ATP and the quaternized nitrogen of the dimethylamino propyl side chain of chlorpromazine. Surface potential measurements of chlorpromazine-ATP solutions as a function of pH indicated the presence of two distinct complexes, with one in the pH range of about 6.5-7.5 and the other forming below pH 5.5.
The wide variety of biochemical alld physiological effects produced by chlorpromazine (CPZ) has made difficult the development of the molecular basis for the action of this drug. Most of the biochemical studies reported up to now seem t,o point to the action of CPZ on systems which are involved in phosphorylation reactions (1). Strong evidence has been presented (2, 3) for t’he inhibition of phosphorylating syst’ems through the mechanism of complex formation bet’ween phenothiazine and coenzyme, e.g., purinc or pyrimidine nucleotide, of the particular apocnayme under study. In addition to its biochemical characteristics, the surface activity of the phenothiazines has been recognized for some t,irne (-1, 3) and quite recenl Iy was the subject of an cxtcnsive report (G). The biochemical evidence for the with int,eraction of the phenothiazines nucleotides such as adenosine triphosphat,e (ATP), coupled with the dcmonstrat,ed surface activity of the phenothiazines, indicates that one aspect of the mechanism of act’ion of these drugs in biological systems may involve
the forlllation
of a conlplex
surface active. On the basis of these considerations,
mhich
studies of the surface chemical properties of solutions of CPZ in the presence of ATP were made. As a result of this work, evidence of specific complex formation between CPZ and ATP was found. This report is an account of some characteristics of the complex formed between CPZ and ATP. EXPERIMENTAL
PROCEDURE
Surjace tension measurements. Surface tensions of solutions were measured by the Wilhelmy hanging-plate technique. The apparatus used employed a sandblasted platinum blade and a double-beam torsion balance. The torsion balance was adjusted to read zero with the wettable plate suspended in the air. The balance was then lowered carefully until the plate just contacted the liquid surface. The plate was immediately wetted and pulled into the bulk of the liquid. The force per unit length acting on the blade was then measured by restoring the plate to its initial position. Swface potential mea.surements. Surface or T’olta potentials of solutions of CPZ and CPZATP mixtures were measured by the ionizing air electrode technique (7). A Iieithley model 610 electrometer was used to directly measure the phase boundary potentials of clean water surfaces and surfaces of CPZ solutions. The electrometer was incorporated into a circuit which had a Beckman glass electrode-8g/AgCl-electrode as refer-
is
some 321
(CHLORPROMAZINE) X lo4 Fro. 1. Effect of adenosine triphosphate on the surface tensions of chlorpromazine solutions at pH 3.0. Solutions unbuffered; yH adjusLed with HCl. Concentrations (Mj of ATP: A none; IZi---II 1 X 1c4; X-X 2 X IOV; O----O 3 X lo-*; X single esperiAments. 72.0
4x 10-4 M
68.0 5X 10-4 M
Mg ADDED
1 10 x 10-d Y
56.0
52.0
v 100% ATP
[ 1
20
40
60 % CPZ
80
100 0% cATP1
FIG. 2. Surface tensions of chlorpromazine-adenosine triphosphate mixtures. The tatal molar concentrat,ions were held constant and the percent.age of chlorpromazine was varied. The initial values of chlorpromazine concentIration are listed on the right-hand ordinate.
COMPLEX
FORMATION
BETWEEN
CHLORPROMAZINE
encc, with the Ag/AgCl electrode connected t,o the electrometer ground terminal. The circuit was completed with a radioactive probe containing Razz6positioned about 1 cm from the interface. This arrangement also permitted the measurement of pH using the Keithley electrometer as pH meter. Solutions were prepared at concentrations of 5 X 10d4 M (CPZ), 5 X lo-” M (ATP), and 1OW~11salts as KC1 or KC1 with HCl (acid solutions) or KC1 with KOH (basic solutions). Materials. The chlorpromazine and diphenylpyraline (Hispril) used in these studies was gencrously dona,ted by Smith, Kline and French, Inc.; lysergic acid diethylamide was purchased from Sandoz Pharmaceuticals. These were purified by recrystallization and three extractions with ethyl ether to remove surface-active impurities. Adenosine triphosphatc was purchased from Calbiothem and used without further purification. All salts used were of reagent grade, and solutions were prepared with double-distilled water prepared in an all-quartz still.
1
RESULTS
The effects of ,4TP on the surface tension of CPZ solutions at pH 7 are illustrated in Fig. 1. Here the surface tensions of solutions of these materials are plot.ted against the logarit,hm of the CPZ concentrat,ion. The curve for CPZ alone is at t,he far right,. Upon t,he addit,ion of ATP t’o CPZ solutions at pH 7, the surface tension-log concentration curve undergoes a shift t.o lower concentrations of CPZ. In addition, t,he maximum surface-tension lowering is a function of the ATP concentration, occurring at pH 7 at about 3 X 1O-4 M, ATP, and 7 X 1O-4 M, CPZ. The presence of ATP in solut’ions of lysergic acid diethylamide, and Hispril causes a similar decrease in surface tension. Surface tensions of ATP-CPZ mixt’ures as a function of mole per cent of dissolved solids at three different CPZ concentrations are shown in lcig. 2. A minimum in surface tension is evident,, and appears at, about the same mole per cent at, each concentrat’ion of CPZ and ATP. The addition of Mg++ t’o an ATP-CPZ lnisture reduced the surface activity of the complex as evidenced by the experiments in Fig. 2 labeled with arrows. Figure 3 shows the effects of variation of nucleotidc structure on the surface tension of CPZ solut,ions. Here the surface tensions of two concentratjions of CPZ are plotted vs. a dimensionless abscissa. The abscissa relates the surface tension to t,he nucleotide used,
AND ATP
323
I
1
I
I
ADENOSINE
AMP
ADP
ATP
NUCLEOTIDE
3. Effect of variation of nucleotide structure on the surface tension of chlorpromazine solutions at pH 3.0. Chlorpromazine concentration: upper curve, 2 X 10m4M; lower curve, 5 X lO+ M. Nucleotide concentrations, 8 X lo-* M. FIG.
e.g., adenosine, adenosine monophosphate (AMP), etc. Note that t’he surface tension of these CPZ solutions is insignificantly altered by adenosine and AMP. It is only when ADP and ATP are added that the surface tension is changed, and most markedly by ATP. Figure 4 shows the surface potentials of solut8ions of CPZ alone (ordinate to the right) and CPZ plus ATP (ordinate t’o the left) as a function of pH. Upon addition of ATP to CPZ solutions at acid values of pH, t’he surface potential increased immediately to a new value. This value remained constant as the pH increased up t’o 5.5. Then, in contrast, to the behavior of the CPZ alone, there was a change in surface potential to a new constant, value greater t’han that of the value of surface potential of CPZ in the presence of ATP below pH 5.5. This second process appeared to have an inflection point at about pH 6.3, probably corresponding to the titration of the ring nitrogen of CPZ (carbazole pK = 6.4). The surface potential in condensed or concentrated surface films is principally a function of dipole moment
324
BLEI
6
7
6
9
PH
FIG. 4. Effect of pH on the surface potentials, AV in millivolts, of solutions of chlorpromazine alone (upper curve, ordinates to the right) and in the presence of adenosine triphosphate (lower curve, ordinate to the left). Solutions contained 5 X 10e4 11f CPZ; 5 X 10e4 IIf ATP; and 1 X 10d2 M salt as KC1 or KC1 plus HCl [acid solutions, and KC1 plus KOH (alkaline solutions)].
and the electrical potential of the double layer. It would therefore appear that the presence of ATP in CPZ solutions in some way alt#ered either or both of these properties. SUMMARY
The surface t’ension of solutions of CPZ is markedly lowered in the presence of ATP, which is itself not surface active. There is a maximum in the surface tension change demon&rated by varying the ratios of (CPZ)/(ATP) over a range of concentrations of these substances from 1 to 10 X 10e4 M. Studies on t’he effects of changing nucleot’ide struct’ure indicated that the chain length of the pyrophosphate moiety is critical and that salt formation between the ATP phosphate and tail nitrogen of CPZ may be a principal driving force for the reaction. Surface potential measurements indicated the presence of an apparent pK at, pH 6.3 in ATP-CPZ mixtures which was not observed in pure CPZ solutions.
ACKNOWLEDGMENT
The author wishes to acknowledge the valuable technical contributions of Mr. J. S. Wortham and Mr. C. Herrmann. REFERENCES 1. MASURAT, T., GREESBERG, S. ?\I., RICE, E. G., HERKDON, J. F., ASD VAN LOON, E. J., Biochem. Pharmacol. 6, 20 (1960) and refcrcnccs therein. Ii., OZAWA, T., AND NAGATSU, T., 2. YAM, Biochem. Biophys. Acta 43, 310 (1960). 3. CARVEIL, M. J., Biochem. Phar?nacol. 12, 19 (1963). 4. SCHOLTAN, W., Kolloid-Z. 142, 84 (1955). 5. VILLALONGA, F., FRIED, E., AND IZQUIEBDO, J. A., Arch. Intern. Pharmacoclyn. 130, 2G0 (1961). 6. SEEMA~-, P. M., ASD BIALY, II. s., Biochem. Pharmacol. 12, 1181 (1963). 7. ALEXANDEIL, A.E., ANDJOHSSOX, P., “Colloid Science.” Oxford Univ. Press, London and New York (1950).
OF
BIOCHEMISTRY
Complex
AND
Formation
BIOPHYSICS
321-324 (1965)
109,
between
Chlorpromazine
and
Adenosine
Triphosphate IRA BLEI Research Division,
Melpar,
Received
Inc., Falls
September
Church,
Virginia
2, 1964
The surface tensions of solutions of chlorpromazine have been shown to decrease in the presence of ATP. The data indicate reversible formation of a complex of greater surface activity than chlorpromazine alone. The extent of surface chemical complex formation of chlorpromazine with homologs of ATP of shorter phosphate chain length indicated that the reaction with ATP was specific and probably was a result of salt formation between the phosphate groups of ATP and the quaternized nitrogen of the dimethylamino propyl side chain of chlorpromazine. Surface potential measurements of chlorpromazine-ATP solutions as a function of pH indicated the presence of two distinct complexes, with one in the pH range of about 6.5-7.5 and the other forming below pH 5.5.
The wide variety of biochemical alld physiological effects produced by chlorpromazine (CPZ) has made difficult the development of the molecular basis for the action of this drug. Most of the biochemical studies reported up to now seem t,o point to the action of CPZ on systems which are involved in phosphorylation reactions (1). Strong evidence has been presented (2, 3) for t’he inhibition of phosphorylating syst’ems through the mechanism of complex formation bet’ween phenothiazine and coenzyme, e.g., purinc or pyrimidine nucleotide, of the particular apocnayme under study. In addition to its biochemical characteristics, the surface activity of the phenothiazines has been recognized for some t,irne (-1, 3) and quite recenl Iy was the subject of an cxtcnsive report (G). The biochemical evidence for the with int,eraction of the phenothiazines nucleotides such as adenosine triphosphat,e (ATP), coupled with the dcmonstrat,ed surface activity of the phenothiazines, indicates that one aspect of the mechanism of act’ion of these drugs in biological systems may involve
the forlllation
of a conlplex
surface active. On the basis of these considerations,
mhich
studies of the surface chemical properties of solutions of CPZ in the presence of ATP were made. As a result of this work, evidence of specific complex formation between CPZ and ATP was found. This report is an account of some characteristics of the complex formed between CPZ and ATP. EXPERIMENTAL
PROCEDURE
Surjace tension measurements. Surface tensions of solutions were measured by the Wilhelmy hanging-plate technique. The apparatus used employed a sandblasted platinum blade and a double-beam torsion balance. The torsion balance was adjusted to read zero with the wettable plate suspended in the air. The balance was then lowered carefully until the plate just contacted the liquid surface. The plate was immediately wetted and pulled into the bulk of the liquid. The force per unit length acting on the blade was then measured by restoring the plate to its initial position. Swface potential mea.surements. Surface or T’olta potentials of solutions of CPZ and CPZATP mixtures were measured by the ionizing air electrode technique (7). A Iieithley model 610 electrometer was used to directly measure the phase boundary potentials of clean water surfaces and surfaces of CPZ solutions. The electrometer was incorporated into a circuit which had a Beckman glass electrode-8g/AgCl-electrode as refer-
is
some 321
(CHLORPROMAZINE) X lo4 Fro. 1. Effect of adenosine triphosphate on the surface tensions of chlorpromazine solutions at pH 3.0. Solutions unbuffered; yH adjusLed with HCl. Concentrations (Mj of ATP: A none; IZi---II 1 X 1c4; X-X 2 X IOV; O----O 3 X lo-*; X single esperiAments. 72.0
4x 10-4 M
68.0 5X 10-4 M
Mg ADDED
1 10 x 10-d Y
56.0
52.0
v 100% ATP
[ 1
20
40
60 % CPZ
80
100 0% cATP1
FIG. 2. Surface tensions of chlorpromazine-adenosine triphosphate mixtures. The tatal molar concentrat,ions were held constant and the percent.age of chlorpromazine was varied. The initial values of chlorpromazine concentIration are listed on the right-hand ordinate.
COMPLEX
FORMATION
BETWEEN
CHLORPROMAZINE
encc, with the Ag/AgCl electrode connected t,o the electrometer ground terminal. The circuit was completed with a radioactive probe containing Razz6positioned about 1 cm from the interface. This arrangement also permitted the measurement of pH using the Keithley electrometer as pH meter. Solutions were prepared at concentrations of 5 X 10d4 M (CPZ), 5 X lo-” M (ATP), and 1OW~11salts as KC1 or KC1 with HCl (acid solutions) or KC1 with KOH (basic solutions). Materials. The chlorpromazine and diphenylpyraline (Hispril) used in these studies was gencrously dona,ted by Smith, Kline and French, Inc.; lysergic acid diethylamide was purchased from Sandoz Pharmaceuticals. These were purified by recrystallization and three extractions with ethyl ether to remove surface-active impurities. Adenosine triphosphatc was purchased from Calbiothem and used without further purification. All salts used were of reagent grade, and solutions were prepared with double-distilled water prepared in an all-quartz still.
1
RESULTS
The effects of ,4TP on the surface tension of CPZ solutions at pH 7 are illustrated in Fig. 1. Here the surface tensions of solutions of these materials are plot.ted against the logarit,hm of the CPZ concentrat,ion. The curve for CPZ alone is at t,he far right,. Upon t,he addit,ion of ATP t’o CPZ solutions at pH 7, the surface tension-log concentration curve undergoes a shift t.o lower concentrations of CPZ. In addition, t,he maximum surface-tension lowering is a function of the ATP concentration, occurring at pH 7 at about 3 X 1O-4 M, ATP, and 7 X 1O-4 M, CPZ. The presence of ATP in solut’ions of lysergic acid diethylamide, and Hispril causes a similar decrease in surface tension. Surface tensions of ATP-CPZ mixt’ures as a function of mole per cent of dissolved solids at three different CPZ concentrations are shown in lcig. 2. A minimum in surface tension is evident,, and appears at, about the same mole per cent at, each concentrat’ion of CPZ and ATP. The addition of Mg++ t’o an ATP-CPZ lnisture reduced the surface activity of the complex as evidenced by the experiments in Fig. 2 labeled with arrows. Figure 3 shows the effects of variation of nucleotidc structure on the surface tension of CPZ solut,ions. Here the surface tensions of two concentratjions of CPZ are plotted vs. a dimensionless abscissa. The abscissa relates the surface tension to t,he nucleotide used,
AND ATP
323
I
1
I
I
ADENOSINE
AMP
ADP
ATP
NUCLEOTIDE
3. Effect of variation of nucleotide structure on the surface tension of chlorpromazine solutions at pH 3.0. Chlorpromazine concentration: upper curve, 2 X 10m4M; lower curve, 5 X lO+ M. Nucleotide concentrations, 8 X lo-* M. FIG.
e.g., adenosine, adenosine monophosphate (AMP), etc. Note that t’he surface tension of these CPZ solutions is insignificantly altered by adenosine and AMP. It is only when ADP and ATP are added that the surface tension is changed, and most markedly by ATP. Figure 4 shows the surface potentials of solut8ions of CPZ alone (ordinate to the right) and CPZ plus ATP (ordinate t’o the left) as a function of pH. Upon addition of ATP to CPZ solutions at acid values of pH, t’he surface potential increased immediately to a new value. This value remained constant as the pH increased up t’o 5.5. Then, in contrast, to the behavior of the CPZ alone, there was a change in surface potential to a new constant, value greater t’han that of the value of surface potential of CPZ in the presence of ATP below pH 5.5. This second process appeared to have an inflection point at about pH 6.3, probably corresponding to the titration of the ring nitrogen of CPZ (carbazole pK = 6.4). The surface potential in condensed or concentrated surface films is principally a function of dipole moment
324
BLEI
6
7
6
9
PH
FIG. 4. Effect of pH on the surface potentials, AV in millivolts, of solutions of chlorpromazine alone (upper curve, ordinates to the right) and in the presence of adenosine triphosphate (lower curve, ordinate to the left). Solutions contained 5 X 10e4 11f CPZ; 5 X 10e4 IIf ATP; and 1 X 10d2 M salt as KC1 or KC1 plus HCl [acid solutions, and KC1 plus KOH (alkaline solutions)].
and the electrical potential of the double layer. It would therefore appear that the presence of ATP in CPZ solutions in some way alt#ered either or both of these properties. SUMMARY
The surface t’ension of solutions of CPZ is markedly lowered in the presence of ATP, which is itself not surface active. There is a maximum in the surface tension change demon&rated by varying the ratios of (CPZ)/(ATP) over a range of concentrations of these substances from 1 to 10 X 10e4 M. Studies on t’he effects of changing nucleot’ide struct’ure indicated that the chain length of the pyrophosphate moiety is critical and that salt formation between the ATP phosphate and tail nitrogen of CPZ may be a principal driving force for the reaction. Surface potential measurements indicated the presence of an apparent pK at, pH 6.3 in ATP-CPZ mixtures which was not observed in pure CPZ solutions.
ACKNOWLEDGMENT
The author wishes to acknowledge the valuable technical contributions of Mr. J. S. Wortham and Mr. C. Herrmann. REFERENCES 1. MASURAT, T., GREESBERG, S. ?\I., RICE, E. G., HERKDON, J. F., ASD VAN LOON, E. J., Biochem. Pharmacol. 6, 20 (1960) and refcrcnccs therein. Ii., OZAWA, T., AND NAGATSU, T., 2. YAM, Biochem. Biophys. Acta 43, 310 (1960). 3. CARVEIL, M. J., Biochem. Phar?nacol. 12, 19 (1963). 4. SCHOLTAN, W., Kolloid-Z. 142, 84 (1955). 5. VILLALONGA, F., FRIED, E., AND IZQUIEBDO, J. A., Arch. Intern. Pharmacoclyn. 130, 2G0 (1961). 6. SEEMA~-, P. M., ASD BIALY, II. s., Biochem. Pharmacol. 12, 1181 (1963). 7. ALEXANDEIL, A.E., ANDJOHSSOX, P., “Colloid Science.” Oxford Univ. Press, London and New York (1950).