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spectrophotometric titrator
ANALYTICAL
A
21,
BIOCHEMIS’IWY
Recording
401410
PotentiometriclSpectrophotometric JOHN
Division
of Biochemistry, and
(1967)
Department
Titrator
E. O’HAGAN
Department of Pathology, Princess Alesandra Hospital, of Biochemistry, University of Queensland, Brisbane, Queensland, Australia Received
March
23, 1967
In general, studies of the interactions of smaller molecules with proteins are performed most satisfactorily under equilibrium conditions. However, this approach was found to be not entirely reproducible and also time-consuming when applied to the study of the attachment of nickel mesoporphyrin (as a substitute for heme) to apohemoglobin and apomyoglobin, using the technique reported by O’Hagan (1). To overcome these difficulties, an instrument was developed by combining, through a specially constructed titration cell, a Radiometer titrator/ titrigraph/syringe buret system with a Shimadzu automatic recording spectrophotometer. Using the technique to be described, it is possible to obtain a complete pH titration curve on the protein complexes in about one hour, with an accuracy suitable for most purposes. Some preliminary results with the technique have already been report,ed by O’Hagan and Moore (3). MATERIALS
AND
EQUIPMEXT
Substances used to check the suitability
of the equipment were: tryst., lyophilized, salt-free. Batch 2. Seravac Laboratories Pty. Ltd. Willow Road, Colnbrook, England. This and the following material was found to be in the ferric state. Myoglobin (equine) 1X tryst., lyophilized, salt-free. Batch 17. Sernvac Laboratories. Niclcel mesoporphyrin was prepared by the method of O’Hagan (1). Human apohemoglobin was prepared by the method described by Rossi Fanelli, Antonini, and Caputo (2) as modified by O’Hagan (1) and its concentration measured by titration with heme. A stock solution of the “straight-line” buffer of Ellis (4) which was 0.1 M with respect to sodium carbonate, 2-amino-2-methyl-1,3-propanediol, sodium dihydrogen phosphate, and citric acid, was used for making all solutions for titration.* Myoglobin
(whale skeletal muscle) lx
401
402
JOHN
E.
O’HAGAN
Referewe buffers: 0.05 111potassium hydrogen phthalate was prepared from British Drug Houses Laboratory Reagent, certified by the National Chemical Laboratory, London, to have a pH of 4.00 at 20” at this concentration and was used as a primary standard. 0.01 111 sodium borate (pH 9.225 at 20”) was used as a secondary standard to check the spread of the pH recording system. Instruments: Radiometer (Copenhagen) titrator type TTTlc, with titrigraph type SBR 2c and syringe buret type SBU Ia. Radiometer semimicro glass electrode G222B (pH range O-14) and calomel electrode K401 were connected to the titrator and inserted into holders on a Plexiglas frame. This frame was attached by means of a knurled thumb screw to the constant-temperature cuvet holder of the spectrophotometer. The holder had been modified to accept 4 cm path length cuvets (Hellma No. 100 OS) and had a screw to fix it firmly to its base plate after alignment. Several stirring systems were tried-magnetic, gas bubbling, propellor types, and combinations of these-but t,hat most satisfactory for the purpose was constructed as follows: One end of a 3 mm i.d. Pyrex glass tube 3 cm long was sealed in a flame and flattened over about 1 cm and this section given a slight longitudinal twist. The other end was fixed with sealing wax over the shaft of a tiny motor (TKK 15 Mabuchi motor, Tokyo Kagaku K.K., Tokyo, Japan). The motor was mounted on the Plexiglas frame between, and in line with, the two electrodes. The tip of a 6 cm polythene tube of 1.5 mm o.d. was constricted slightly by carefully and rapidly warming in a very small flame a section at about 3 cm from the end, stretching this a little, cooling while stretched, and cutting at the constriction with a scalpel blade. The resulting hole was about 0.3 mm in diameter. The other end of the tube was connected to the syringe buret and the tip of the tube was fixed just opposite the middle of the bulb of the glass electrode. The positioning of the tube was critical for optimal results and was made by trial and error once the system was functioning. The stirrer motor was connected to a small box containing a switch, a 10 ohm rheostat, O-2 V voltmeter, and connections to a 2.0 V accumulator. In operation, the rheostat was turned forward to start the motor and then back to a point (in our case 0.7 V) at which there was adequate stirring without bubbles being introduced. Such a procedure was necessary to overcome the starting torque of the motor. A new cuvet compartment cover was constructed with top 12 cm higher than the standard one, t.o accommodate the electrodes, and holes were provided in the top to pass the electrode leads, stirrer leads, and the polythene tube. After insertion of the leads and tube, the holes were sealed with wax. The inside of the cover was painted with flat black paint and the system tested to ensure that no extraneous light entered.
COMBINED
TITRATION
SYSTEM
403
A water bath equipped with a Thermomix II thermoregulator (B. Braun, Melsungen) provided a flow of water at constant temperature for the jacket of the cuvet holder. The layout of the equipment is shown in Figures 1 and 2 and close-ups of the cuvet holder with electrodes, stirrer, and polythene tube are given in Figures 3 and 4.
FIG. 1. General view of combined titration apparatus. The titrigraph/titrator/ syringe buret system and constant-temperature water bath are on the rear bench; the titration cell with electrodes and stirrer are in the cuvet holder compartment of the spectrophotometer. The compartment cover has been let back; it is brought forward and downward during the titration. After this picture was taken a voltmeter was added to the stirrer control unit and the fine polythene tube from the buret replaced the heavier polyvinyl chloride tube shown here.
To operate the equipment, the titration apparatus was switched on and adjusted, the cuvet was filled with 12 ml of the phthalate reference buffer and placed in the holder, and the Plexiglas frame holding the electrodes and stirrer was screwed down on top of the holder. With holder assembly outside the cuvet compartment of the spectrophotometer, the stirrer was switched on and adjusted to a point where there was maximum stirring without air bubbles being sucked down into the liquid. The voltage required for this was noted, and after starting the mot’or the rheostat was set to give this voltage on all subsequent runs. After setting
404
FIG.
JOHX
2. Schematic
diagram
showing
E.
O’HAGAS
interconnections
of components
of the
system.
the pH scale with the motor running, the electrodes were washed and the spread of the scale was checked against the sodium borate reference buffer. The best position for the outlet of the polythene tube relative to the
FIG. 3. Close-up of the titration cell showing glass and calomel electrodes, stirrer motor, clamping nut, delivery tube, thermostatted cuvet holder, and compartment cover. Only the two middle sections of the four-section holder are used. Connections from the thermostat have brrn removed.
COMBINED
TITRATIOK
SYSTEM
405
FIG. 4. Underside of cuvet cover showing position of glass electrode, polythene tube and mounting, stirrer, calomel electrode, and hole for locking screw.
glass electrode was determined, using the standard method for checking the t,itrimet,er described in the Radiometer instruction manual, i.e., by titrating a sodium carbonate solution with hydrochloric acid. The position of the outlet was varied by shifting a small Plexiglas holder, into which it was inserted, relative to the bulb of the glass electrode. Careful adjustment permitted the titration to proceed with the shortest time constant for the system and t,his gave a curve on the titrigraph which approached a thin smooth line. Another tube to introduce an inert gas was an opt,ional addition, RESULTS
Titration of Fewimyoglobins. To check the suitability of the apparatus for the study of ionizations in hemoproteins, the dissociations of the ironbound water molecules in sperm whale and horse ferrimyoglobins were examined. George and Hanania (5) had found the ionization Fe+ CHTO) e FeOH
+ H’
406
JOHN
E.
O’HAGAN
in horse ferrimyoglobin to have, in borate buffer at 2O”C, a pK’ of 8.89 -+- 0.01, within the ionic strength range 0.004-0.009 inclusive. The titration of horse ferrimyoglobin in the pH range 6.6-11.3 was performed as follows: Into the cuvet were placed 12 ml of water, 0.1 ml of the Ellis buffer, and enough dried ferrimyoglobin to give an absorbance at 409.5 rnp of 0.8-1.0 at the starting point of the titration i.e., pH 6.6. It is not essential to know the concentration of ferrimyoglobin but it can be calculated from the molar absorbance coefficient, which is approximately 171 at 409 rnp (6) so that the concentration was about 6 X 10e6M. The absolute value depends on the molecular weight, the exact value of which has yet to be decided upon-see Boardman’s (7) discussion of this matter, and that of Hanania, Yeghiayan and Cameron (8) in respect to sperm whale myoglobin. The syringe buret was filled with 0.11M NaOH, the polythene tube connected and filled, and the tip rinsed with distilled water before the electrode assembly was placed on the cuvet holder. The titration was started and the change in absorbance with time recorded on the spectrophotometer. As the titrigraph reached fixed points on the pH scale (e.g. 7.5, 8.0, 8.5), these were marked on the spectrophotometric titration curve because the relationship between the pH scale and the time scale was not linear. Similar runs were made with sperm whale ferrimyoglobin. The recordings from these titrations are shown in Figure 5 and the plot on a linear scale in Figure 6. From inspection of Figure 6, the pK’ for the dissociation of the iron-bound water molecule in horse ferrimyoglobin in the Ellis buffer (I = 0.007) was found to be 9.0, which compares reasonably well with the value of 8.89 + 0.01 found by George and Hanania (5) in borate buffer (I = 0.004 to 0.009). The corresponding pK’ for the ionization in sperm whale ferrimyoglobin was found to be 9.15. Side-Chain Linkages i?l Nemoproteins. The method was applied to the study of the attachment of nickel mesoporphyrin to apohemoglobins and apomyoglobins and preliminary results have been reported by O’Hagan and Moore (3). As an illust,ration of the type of result possible, the titration of the complex of nickel mesoporphyrin and human apohemoglobin A will be described. To prepare the complex, 1 ml of 2.0 x 1O-4 M apohemoglobin solution in water was pipetted into a 50 ml standard flask, 0.3 ml of the 0.1 Al Ellis buffer added, followed by 1 ml of 1.0 x 1O-4M nickel mesoporphyrin solution in 0.01 M NaOH, and distilled water added to 50 ml. The apohemoglobin was used in considerable excess to allow for the possibility of any noncombining material being present and its molarity was based on the assumption that 1 mole of hematin or nickel mesoporphyrin combined with 1 mole of apohemoglobin (i.e., 1 Alor /3 chain).
COMBINED
TITRATION
407
SYSTEM
Time
8.0
6 1.80 -
Sperm whole myoglobin Fe’+
160 -
FIG. 5. Titration of the ionization of the iron-bound water molecule in horse and in sperm whale ferrimyoglobin using the combined titration system. Details given in text. Numbers on curves indicate pH values marked during the courses of the titrations, each of which took 8 minutes to complete.
0*9O-80-7A O-60.5-
6
I 7
I 8
I 9 PH
I 10
I 11
2
FIG. 6. Plot on linear scale from data from Figure 5 of ionizations bound water molecules in horse (A) and sperm whale ferrimyoglobins
of the iron(a).
408
JOHN
E.
O’HAGAX
Into the cuvet of the titration apparatus was placed 12.5 ml of the mixture and t.he titration commenced using 0.1 M HCl, 0.1 JI NaOH, or 2.5 M NaOH (above pH 10). The result of the three titrations combined is shown in Figure 7. This curve represents the increase in absorbance
FIG. 7. Attachment of nickel mesoporphyrin to human adult apohemoglobin. curve represents a plot on a linear scale of three pairs of titrations-with HCl, 0.1 M NaOH, and 2.5 M NaOH-and indicates the absorbance increment produced in the nickel mesoporphyrin solution by the apohcmoglobin.
This 0.1-M
(Ai)
(Ai) obtained on adding the apohemoglobin, that is, it has been obtained by subtracting the results of the titration under identical conditions of a solution of nickel meeoporphyrin alone. DISCUSSION
The technique has proved very useful in performing spectrophotometric titrations on a series of complexes of nickel mesoporphyrin with apohemoglobins and apomyoglobins. For this purpose, since it was mainly a comparison between the curves obtained with apoproteins from different species that was required, it was immaterial whether each point on the titration curve represented the final equilibrium position. The main advantage over previous techniques was that the curves were reproducible and allowed definite comparisons to be made between the combining groups in the various species examined. The technique is rapid and convenient, in use, although methods such as those described by Zimmer and Reinert (9) are probably of slightly greater accuracy.
COMBINED
TITRATION
SYSTEM
409
The accuracy of the present equipment could be improved by running the titrigraph recorder at a slower speed. The work described here was run on the 27 min range of the recorder, i.e., with chart motor B at 4 rpm, pen motor C at 30 rpm, and chart gear A 2.5 mm/rev, pen gear D l%/ rev. The recorder will operate over twelve speed ranges up to 35 hr for a 25 cm chart travel and 100% syringe travel. By using one of the intermediate ranges (e.g., 2 or 3.5 hr) , conditions closer to those of equilibrium would result. However, under these circumstances it may be necessary to use a finer gage of polythene tube to prevent excessive leaking of the titrant solution. In this respect, it is important that the clip of the syringe buret grips the plunger firmly (but not tightly) to prevent plunger creeping, due to the tendency of the titrant solution to syphon. The system would appear to offer advantages over that of Zimmer and Reinert (9) and of French and Bruice (10) in that both spectrophotomet,ric and potentiometric recordings are made, and corrections for the change in volume on addition of the titrant solution can be allowed for, if necessary, by measurements from the titrigraph chart. The Radiometer system has been so designed that the titrant solution is added slowly where the pH is changing rapidly and this has obvious advantages. Future Developments. The system could be improved further by using the relay system of the titrigraph driving the chart to operate the time drive of the spectrophotometer recorder, so that a linear recording of absorbance versus pH could be obtained. Since the operation is performed at a fixed wavelength, the fully recording spectrophotometer is not required and it should be possible to use a manual spectrophotometer coupled to a linear-log recorder to obtain an S-E’ plot. This would give a more compact instrument and the recording spectrophotometer would be released for other work. Such a linear plot would permit a more accurate determination of the pK’ because it would allow the drawing of the first (AA/APH) and second (A”A/A~H~) derivative curves, as described by Vogel (11). The lack of linearity does not make it possible to do this with sufficient accuracy in the experiments described above, and the pKt has been obtained in each case by drawing a tangent to the curve. SUMMARY A system for the simultaneous recording of spectrophotometric and potentiometric titrations at constant temperature is described. Application to the determination of the pK’ of the ionization of the iron-bound water molecule in horse and in sperm whale ferrimyoglobin and to the study of the attachment of nickel mesoporphyrin to human apohemoglobin A is given.
410
JOHN
E. O’HAGAN
ACKNOWLEDGMENTS I am indebted to the National Heart Foundation of Australia for a Grant-in-Aid and for the Radiometer equipment, and to the National Health and Medical Research Council of Australia for a Research Fellowship. I am grateful to Professor E. C. Webb and Drs. 0. W. Powell and R. A. Caldwell for encouragement, to Mrs. P. J. Moore for technical assistance, and to Mr. L. Otto for making the modification to the constant-temperature cuvet holder. REFERENCES 1. O’HAGAN, J. E., in “Haematin Enzymes” (Falk, J. E., Lemberg, R., and Morton, R. K., eds.), p. 173. Pergamon Press, Oxford, 1961. 2. ROSSI FANELLI, A., ANTONINI, E., AND CAPUTO, A., Biochim. Biophys. Acta 28, 221 (19.58). 3. O’HAGAN, J. E., AND MOORE, P. J., Proc. Intern. Sump. Comparative Hemoglobin Structure, Thessaloniki, Greece, April, 1966, p. 83. 4. ELLIS, D. A., Nature 191, 1099 (1961). 5. GEORGE, P., AND HANANIA, G. I. H., B&hem. J. 52,517 (1952). 6. THEORELL, H., AND AKESON, A., Ann. Acad. Sci. Fennicae Ser. A. ZZ, No. 60, 303 ( 1955). 7. BOARDMAN, N. K., in “Haematin Enzymes” (Falk, J. E., Lemberg, R., and Morton, R. K., eds.), p. 140. Pergamon Press, Oxford, 1961. 8. HANANIA, G. I. H., YEGHUYAN, A., AND CAMERON, B. F., Biochem. .Z. 98, 189 (1966). 9. ZIMMER, C., AND REINERT, H., Anal. Biochesm. 14, 1 (1966). 16. FRENCH, T. C., AND BRUICE, T. C., Biochemistry 3, 1539 (1964). Book of Quantitative Inorganic Analysis, Including EleIl. VOGAL, A. I., “A Text 3rd ed., p. 931. Longmans, London, 1962. mentary Instrumental Analysis,”
A
21,
BIOCHEMIS’IWY
Recording
401410
PotentiometriclSpectrophotometric JOHN
Division
of Biochemistry, and
(1967)
Department
Titrator
E. O’HAGAN
Department of Pathology, Princess Alesandra Hospital, of Biochemistry, University of Queensland, Brisbane, Queensland, Australia Received
March
23, 1967
In general, studies of the interactions of smaller molecules with proteins are performed most satisfactorily under equilibrium conditions. However, this approach was found to be not entirely reproducible and also time-consuming when applied to the study of the attachment of nickel mesoporphyrin (as a substitute for heme) to apohemoglobin and apomyoglobin, using the technique reported by O’Hagan (1). To overcome these difficulties, an instrument was developed by combining, through a specially constructed titration cell, a Radiometer titrator/ titrigraph/syringe buret system with a Shimadzu automatic recording spectrophotometer. Using the technique to be described, it is possible to obtain a complete pH titration curve on the protein complexes in about one hour, with an accuracy suitable for most purposes. Some preliminary results with the technique have already been report,ed by O’Hagan and Moore (3). MATERIALS
AND
EQUIPMEXT
Substances used to check the suitability
of the equipment were: tryst., lyophilized, salt-free. Batch 2. Seravac Laboratories Pty. Ltd. Willow Road, Colnbrook, England. This and the following material was found to be in the ferric state. Myoglobin (equine) 1X tryst., lyophilized, salt-free. Batch 17. Sernvac Laboratories. Niclcel mesoporphyrin was prepared by the method of O’Hagan (1). Human apohemoglobin was prepared by the method described by Rossi Fanelli, Antonini, and Caputo (2) as modified by O’Hagan (1) and its concentration measured by titration with heme. A stock solution of the “straight-line” buffer of Ellis (4) which was 0.1 M with respect to sodium carbonate, 2-amino-2-methyl-1,3-propanediol, sodium dihydrogen phosphate, and citric acid, was used for making all solutions for titration.* Myoglobin
(whale skeletal muscle) lx
401
402
JOHN
E.
O’HAGAN
Referewe buffers: 0.05 111potassium hydrogen phthalate was prepared from British Drug Houses Laboratory Reagent, certified by the National Chemical Laboratory, London, to have a pH of 4.00 at 20” at this concentration and was used as a primary standard. 0.01 111 sodium borate (pH 9.225 at 20”) was used as a secondary standard to check the spread of the pH recording system. Instruments: Radiometer (Copenhagen) titrator type TTTlc, with titrigraph type SBR 2c and syringe buret type SBU Ia. Radiometer semimicro glass electrode G222B (pH range O-14) and calomel electrode K401 were connected to the titrator and inserted into holders on a Plexiglas frame. This frame was attached by means of a knurled thumb screw to the constant-temperature cuvet holder of the spectrophotometer. The holder had been modified to accept 4 cm path length cuvets (Hellma No. 100 OS) and had a screw to fix it firmly to its base plate after alignment. Several stirring systems were tried-magnetic, gas bubbling, propellor types, and combinations of these-but t,hat most satisfactory for the purpose was constructed as follows: One end of a 3 mm i.d. Pyrex glass tube 3 cm long was sealed in a flame and flattened over about 1 cm and this section given a slight longitudinal twist. The other end was fixed with sealing wax over the shaft of a tiny motor (TKK 15 Mabuchi motor, Tokyo Kagaku K.K., Tokyo, Japan). The motor was mounted on the Plexiglas frame between, and in line with, the two electrodes. The tip of a 6 cm polythene tube of 1.5 mm o.d. was constricted slightly by carefully and rapidly warming in a very small flame a section at about 3 cm from the end, stretching this a little, cooling while stretched, and cutting at the constriction with a scalpel blade. The resulting hole was about 0.3 mm in diameter. The other end of the tube was connected to the syringe buret and the tip of the tube was fixed just opposite the middle of the bulb of the glass electrode. The positioning of the tube was critical for optimal results and was made by trial and error once the system was functioning. The stirrer motor was connected to a small box containing a switch, a 10 ohm rheostat, O-2 V voltmeter, and connections to a 2.0 V accumulator. In operation, the rheostat was turned forward to start the motor and then back to a point (in our case 0.7 V) at which there was adequate stirring without bubbles being introduced. Such a procedure was necessary to overcome the starting torque of the motor. A new cuvet compartment cover was constructed with top 12 cm higher than the standard one, t.o accommodate the electrodes, and holes were provided in the top to pass the electrode leads, stirrer leads, and the polythene tube. After insertion of the leads and tube, the holes were sealed with wax. The inside of the cover was painted with flat black paint and the system tested to ensure that no extraneous light entered.
COMBINED
TITRATION
SYSTEM
403
A water bath equipped with a Thermomix II thermoregulator (B. Braun, Melsungen) provided a flow of water at constant temperature for the jacket of the cuvet holder. The layout of the equipment is shown in Figures 1 and 2 and close-ups of the cuvet holder with electrodes, stirrer, and polythene tube are given in Figures 3 and 4.
FIG. 1. General view of combined titration apparatus. The titrigraph/titrator/ syringe buret system and constant-temperature water bath are on the rear bench; the titration cell with electrodes and stirrer are in the cuvet holder compartment of the spectrophotometer. The compartment cover has been let back; it is brought forward and downward during the titration. After this picture was taken a voltmeter was added to the stirrer control unit and the fine polythene tube from the buret replaced the heavier polyvinyl chloride tube shown here.
To operate the equipment, the titration apparatus was switched on and adjusted, the cuvet was filled with 12 ml of the phthalate reference buffer and placed in the holder, and the Plexiglas frame holding the electrodes and stirrer was screwed down on top of the holder. With holder assembly outside the cuvet compartment of the spectrophotometer, the stirrer was switched on and adjusted to a point where there was maximum stirring without air bubbles being sucked down into the liquid. The voltage required for this was noted, and after starting the mot’or the rheostat was set to give this voltage on all subsequent runs. After setting
404
FIG.
JOHX
2. Schematic
diagram
showing
E.
O’HAGAS
interconnections
of components
of the
system.
the pH scale with the motor running, the electrodes were washed and the spread of the scale was checked against the sodium borate reference buffer. The best position for the outlet of the polythene tube relative to the
FIG. 3. Close-up of the titration cell showing glass and calomel electrodes, stirrer motor, clamping nut, delivery tube, thermostatted cuvet holder, and compartment cover. Only the two middle sections of the four-section holder are used. Connections from the thermostat have brrn removed.
COMBINED
TITRATIOK
SYSTEM
405
FIG. 4. Underside of cuvet cover showing position of glass electrode, polythene tube and mounting, stirrer, calomel electrode, and hole for locking screw.
glass electrode was determined, using the standard method for checking the t,itrimet,er described in the Radiometer instruction manual, i.e., by titrating a sodium carbonate solution with hydrochloric acid. The position of the outlet was varied by shifting a small Plexiglas holder, into which it was inserted, relative to the bulb of the glass electrode. Careful adjustment permitted the titration to proceed with the shortest time constant for the system and t,his gave a curve on the titrigraph which approached a thin smooth line. Another tube to introduce an inert gas was an opt,ional addition, RESULTS
Titration of Fewimyoglobins. To check the suitability of the apparatus for the study of ionizations in hemoproteins, the dissociations of the ironbound water molecules in sperm whale and horse ferrimyoglobins were examined. George and Hanania (5) had found the ionization Fe+ CHTO) e FeOH
+ H’
406
JOHN
E.
O’HAGAN
in horse ferrimyoglobin to have, in borate buffer at 2O”C, a pK’ of 8.89 -+- 0.01, within the ionic strength range 0.004-0.009 inclusive. The titration of horse ferrimyoglobin in the pH range 6.6-11.3 was performed as follows: Into the cuvet were placed 12 ml of water, 0.1 ml of the Ellis buffer, and enough dried ferrimyoglobin to give an absorbance at 409.5 rnp of 0.8-1.0 at the starting point of the titration i.e., pH 6.6. It is not essential to know the concentration of ferrimyoglobin but it can be calculated from the molar absorbance coefficient, which is approximately 171 at 409 rnp (6) so that the concentration was about 6 X 10e6M. The absolute value depends on the molecular weight, the exact value of which has yet to be decided upon-see Boardman’s (7) discussion of this matter, and that of Hanania, Yeghiayan and Cameron (8) in respect to sperm whale myoglobin. The syringe buret was filled with 0.11M NaOH, the polythene tube connected and filled, and the tip rinsed with distilled water before the electrode assembly was placed on the cuvet holder. The titration was started and the change in absorbance with time recorded on the spectrophotometer. As the titrigraph reached fixed points on the pH scale (e.g. 7.5, 8.0, 8.5), these were marked on the spectrophotometric titration curve because the relationship between the pH scale and the time scale was not linear. Similar runs were made with sperm whale ferrimyoglobin. The recordings from these titrations are shown in Figure 5 and the plot on a linear scale in Figure 6. From inspection of Figure 6, the pK’ for the dissociation of the iron-bound water molecule in horse ferrimyoglobin in the Ellis buffer (I = 0.007) was found to be 9.0, which compares reasonably well with the value of 8.89 + 0.01 found by George and Hanania (5) in borate buffer (I = 0.004 to 0.009). The corresponding pK’ for the ionization in sperm whale ferrimyoglobin was found to be 9.15. Side-Chain Linkages i?l Nemoproteins. The method was applied to the study of the attachment of nickel mesoporphyrin to apohemoglobins and apomyoglobins and preliminary results have been reported by O’Hagan and Moore (3). As an illust,ration of the type of result possible, the titration of the complex of nickel mesoporphyrin and human apohemoglobin A will be described. To prepare the complex, 1 ml of 2.0 x 1O-4 M apohemoglobin solution in water was pipetted into a 50 ml standard flask, 0.3 ml of the 0.1 Al Ellis buffer added, followed by 1 ml of 1.0 x 1O-4M nickel mesoporphyrin solution in 0.01 M NaOH, and distilled water added to 50 ml. The apohemoglobin was used in considerable excess to allow for the possibility of any noncombining material being present and its molarity was based on the assumption that 1 mole of hematin or nickel mesoporphyrin combined with 1 mole of apohemoglobin (i.e., 1 Alor /3 chain).
COMBINED
TITRATION
407
SYSTEM
Time
8.0
6 1.80 -
Sperm whole myoglobin Fe’+
160 -
FIG. 5. Titration of the ionization of the iron-bound water molecule in horse and in sperm whale ferrimyoglobin using the combined titration system. Details given in text. Numbers on curves indicate pH values marked during the courses of the titrations, each of which took 8 minutes to complete.
0*9O-80-7A O-60.5-
6
I 7
I 8
I 9 PH
I 10
I 11
2
FIG. 6. Plot on linear scale from data from Figure 5 of ionizations bound water molecules in horse (A) and sperm whale ferrimyoglobins
of the iron(a).
408
JOHN
E.
O’HAGAX
Into the cuvet of the titration apparatus was placed 12.5 ml of the mixture and t.he titration commenced using 0.1 M HCl, 0.1 JI NaOH, or 2.5 M NaOH (above pH 10). The result of the three titrations combined is shown in Figure 7. This curve represents the increase in absorbance
FIG. 7. Attachment of nickel mesoporphyrin to human adult apohemoglobin. curve represents a plot on a linear scale of three pairs of titrations-with HCl, 0.1 M NaOH, and 2.5 M NaOH-and indicates the absorbance increment produced in the nickel mesoporphyrin solution by the apohcmoglobin.
This 0.1-M
(Ai)
(Ai) obtained on adding the apohemoglobin, that is, it has been obtained by subtracting the results of the titration under identical conditions of a solution of nickel meeoporphyrin alone. DISCUSSION
The technique has proved very useful in performing spectrophotometric titrations on a series of complexes of nickel mesoporphyrin with apohemoglobins and apomyoglobins. For this purpose, since it was mainly a comparison between the curves obtained with apoproteins from different species that was required, it was immaterial whether each point on the titration curve represented the final equilibrium position. The main advantage over previous techniques was that the curves were reproducible and allowed definite comparisons to be made between the combining groups in the various species examined. The technique is rapid and convenient, in use, although methods such as those described by Zimmer and Reinert (9) are probably of slightly greater accuracy.
COMBINED
TITRATION
SYSTEM
409
The accuracy of the present equipment could be improved by running the titrigraph recorder at a slower speed. The work described here was run on the 27 min range of the recorder, i.e., with chart motor B at 4 rpm, pen motor C at 30 rpm, and chart gear A 2.5 mm/rev, pen gear D l%/ rev. The recorder will operate over twelve speed ranges up to 35 hr for a 25 cm chart travel and 100% syringe travel. By using one of the intermediate ranges (e.g., 2 or 3.5 hr) , conditions closer to those of equilibrium would result. However, under these circumstances it may be necessary to use a finer gage of polythene tube to prevent excessive leaking of the titrant solution. In this respect, it is important that the clip of the syringe buret grips the plunger firmly (but not tightly) to prevent plunger creeping, due to the tendency of the titrant solution to syphon. The system would appear to offer advantages over that of Zimmer and Reinert (9) and of French and Bruice (10) in that both spectrophotomet,ric and potentiometric recordings are made, and corrections for the change in volume on addition of the titrant solution can be allowed for, if necessary, by measurements from the titrigraph chart. The Radiometer system has been so designed that the titrant solution is added slowly where the pH is changing rapidly and this has obvious advantages. Future Developments. The system could be improved further by using the relay system of the titrigraph driving the chart to operate the time drive of the spectrophotometer recorder, so that a linear recording of absorbance versus pH could be obtained. Since the operation is performed at a fixed wavelength, the fully recording spectrophotometer is not required and it should be possible to use a manual spectrophotometer coupled to a linear-log recorder to obtain an S-E’ plot. This would give a more compact instrument and the recording spectrophotometer would be released for other work. Such a linear plot would permit a more accurate determination of the pK’ because it would allow the drawing of the first (AA/APH) and second (A”A/A~H~) derivative curves, as described by Vogel (11). The lack of linearity does not make it possible to do this with sufficient accuracy in the experiments described above, and the pKt has been obtained in each case by drawing a tangent to the curve. SUMMARY A system for the simultaneous recording of spectrophotometric and potentiometric titrations at constant temperature is described. Application to the determination of the pK’ of the ionization of the iron-bound water molecule in horse and in sperm whale ferrimyoglobin and to the study of the attachment of nickel mesoporphyrin to human apohemoglobin A is given.
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JOHN
E. O’HAGAN
ACKNOWLEDGMENTS I am indebted to the National Heart Foundation of Australia for a Grant-in-Aid and for the Radiometer equipment, and to the National Health and Medical Research Council of Australia for a Research Fellowship. I am grateful to Professor E. C. Webb and Drs. 0. W. Powell and R. A. Caldwell for encouragement, to Mrs. P. J. Moore for technical assistance, and to Mr. L. Otto for making the modification to the constant-temperature cuvet holder. REFERENCES 1. O’HAGAN, J. E., in “Haematin Enzymes” (Falk, J. E., Lemberg, R., and Morton, R. K., eds.), p. 173. Pergamon Press, Oxford, 1961. 2. ROSSI FANELLI, A., ANTONINI, E., AND CAPUTO, A., Biochim. Biophys. Acta 28, 221 (19.58). 3. O’HAGAN, J. E., AND MOORE, P. J., Proc. Intern. Sump. Comparative Hemoglobin Structure, Thessaloniki, Greece, April, 1966, p. 83. 4. ELLIS, D. A., Nature 191, 1099 (1961). 5. GEORGE, P., AND HANANIA, G. I. H., B&hem. J. 52,517 (1952). 6. THEORELL, H., AND AKESON, A., Ann. Acad. Sci. Fennicae Ser. A. ZZ, No. 60, 303 ( 1955). 7. BOARDMAN, N. K., in “Haematin Enzymes” (Falk, J. E., Lemberg, R., and Morton, R. K., eds.), p. 140. Pergamon Press, Oxford, 1961. 8. HANANIA, G. I. H., YEGHUYAN, A., AND CAMERON, B. F., Biochem. .Z. 98, 189 (1966). 9. ZIMMER, C., AND REINERT, H., Anal. Biochesm. 14, 1 (1966). 16. FRENCH, T. C., AND BRUICE, T. C., Biochemistry 3, 1539 (1964). Book of Quantitative Inorganic Analysis, Including EleIl. VOGAL, A. I., “A Text 3rd ed., p. 931. Longmans, London, 1962. mentary Instrumental Analysis,”