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Analytical scanning isoelectrofocusing
ANALYTICAL
BIOCHEMISTRY
‘%‘i,
Analytical
411426
Scanning
3. Design and Operation
NICHOLAS Laboratory
(1971)
of Protein
Isoelectrofocusing:
of an in Situ Scanning
Apparatus1
CATSIMPOOLAS
Chemistry, Central Soya Research Center, Chicago, Illinois 60639
Received February
24, 1971
The advantage of in situ direct optical scanning techniques in analytical isoelectrofocusing, have been demonstrated both in polyacrylamide gel (1) and sucrose density gradient (2) media. Optical scanning methods have also been used successfully in other analytical separation techniques such as gel chromatography (3), free zone electrophoresis (4)) and density gradient, or semifluid film electrophoresis (5-7). In \situ scanning procedures should be distinguished from other optical scanning methods developed for polyacrylamide gel electrophoresis (8-12) or isoelectrofocusing (13). The latter employ scanning after termination of the experiment and in the absence of current. This imposes imitations in obtaining kinetic data and does not compensate for zone diffusion. A density gradient zone electrophoresis apparatus (14) designed for measuring electrophoretic mobilities incorporates some desirable features that have been adopted in this work but again is not truly “in situ scanning” because the current is interrupted for measurements, and the gradient column is pumped through an ultraviolet flow cell. In view of the suitability and usefulness of the in situ scanning techniques in isoelectrofocusing, electrophoresis, and gel filtration experiments at the analytical level, the development of a scanning apparatus capable of performing these procedures interchangeably was considered desirable. It is the purpose of the present paper to report the basic design of such an apparatus, and, at present, describe its operation in isoelectrofocusing experiments. DESIGN
OF THE
APPARATUS
Overall Description
A schematic diagram of the analytical isoelectrofocusing scanning apparatus (AISA) is shown in Fig. 1. The quartz isoelectrofocusing col’ References 1 and 2 are Parts 1 and 2, respectively, 411
of t&i series.
412
NICHOLAS
CATSIMPOOLAS
Sb
FIG. 1. Schematic diagram of analytical isoelect.rofocusing apparatus (AISA) : Am = ammeter; Cg = column guide ; Cl = cooler; Co = column ; Cp = capillary pH electrode assembly; Cr = circulator (thermostated) ; Eps = electrophoresis power supply; Fd = filling device ; Int = integrator; L = lamp ; Lps = lamp power supply; Ls = leadscrew ; M = monoehromator ; MC = motor control box; Mp = metering pump (or autoburet) ; P = photomultiplier; pH = pH meter; Pm = pump; PO = potentiometer; Pps = photomultiplier power supply; Pt = photometer ; Rc = recorder ; Rs = reservoir ; Sb = standardization buffer; Sm = synchronous motor ; SW = switch; Thp = thermistor probe chamber; Tm = elapsed time indicator; Tp = temperature programmer; Tth = telethermometer; Vc = vacuum pump; Vl = valve; Ws = waste.
(Co) is moved vertically by a synchronous motor (Sm) attached to the leadscrew (Ls). The movement of the motor is controlled by the motor control box (MC). The photomultiplier (P), connected to the photometer (Pt), recorder (Rc), and integrator (Int), detects the absorbance of filtered (M) light from the lamp (L) by the light-absorbing zones of materials in the separating column. The column moves through the column guide (Cg), which bears a slit positioned perpendicularly to the direction of the column movement. The electrophoresis power supply
umn
ANALYTICAL
SCANNING
ISOELECTHOFOCUSISG
413
(Eps) is connected to the electrolyte reservoirs (Rs) . The current is turned on by switches (SW) and is measured by the milli- or microammeters (Am). The duration of isoelectrofocusing is indicated by the elapsed time indicator (Tm). Temperature control for the column is provided by the thermostated water circulator (Cr). For temperatures below ambient, the cooler (Cl) circulates cooling fluid through the coils of the circulator by means of the pump (I’m). Controlled temperature changes can be obtained with the temperature programmer (Tp). The outer jacket of the column is evacuated with the vacuum pump (Vc) in order to avoid condensation of vapors at low cooling temperatures. The temperature of the circulating water is measured in a thermistor probe chamber (Thp) with a telethermometer (Tth). The pH of the column effluent is determined by passing through the capillary pH electrode assembly (Cp) connected to the recorder (Rc). A valve (Vl) is used for introduction of standardization buffer (Sb) into the pH cell. The column is filled from the bottom through a valve (Vl). A photograph of the AISA is shown in Fig. 2. The Column
and Electrolyte
Reservoirs
The Column. Suprasil T-20 (Amersil) material was used for construction of the column which consists of three concentric tubes-inner, middle, and outer corresponding to the separation compartment, cooling jacket, and vacuum jacket (Fig. 3). The outer diameters of these tubes are 4, 9, and 15 mm, and the inner diameters are 3, 7, and 13 mm, respectively. The length of the inner column is approximately 413 mm, with the middle and outer tubes relatively shorter in order to accommodate the water and vacuum outlets as shown in Fig. 4. The ends of the inner tube are connected to the electrolyte reservoirs. The Electrolyte Reservoirs. These were constructed from LKB column adapters (extensive piece, plunger shaft, and clamping collar) cut to appropriate length, and Chromatromix Cheminert fittings (Fig. 4). The extension pieces are glued to the ends of the inner tube of the column and serve as extensions of the column and as a means of screwing the column into t.he plunger shafts. The latter serve as holders of the membrane that separates the electrolyte in the electrolyte reservoir from the electrolyte inside the tube of the shaft. This is accomplished by drilling four holes (in the shape of a cross) perpendicularly to the length of the shaft connecting the outer cylindrical surface of the shaft with the inner tube. The holes are filled with a piece of Styrofoam-cut to the dimensions of the hole-ant1 covered from the outer area with two or three folds of dialysis membrane sheet (Cole-Parmer). The ~~mrhralle is held on the
NICHOLAS
CATSIMPOOLAS
Fro. 2. Photograph of different parts of the AISA: A = telethermometer; B = pH meter; C = reference pH electrode; D = capillary pH electrode assembly in hollow steel box; E = thermistor probe chamber; F = milli- and microammeters (three ranges) ; G = elapsed time indicator ; H = column with upper electrolyte reservoir; I = electrode terminal ; J = three-way valve ; K = electrophoresis power supply; L = digital absorbancemeter (for zeroing baseline) ; M = recorder; N = photometer ; 0 = scanning device ; P = motor control box ; Q = thermostated circulator; R = cooler; S = autoburet ; and T = vacuum pump.
shaft either with 2 O-rings or electrical tape and allows current to pass from the electrolyte in the reservoir to the electrolyte in the tube. As mentioned by Brakke et al. (14) in the description of their density gradient electrophoresis apparatus, the flow of liquid through these membranes
ANALYTICAL
FIG. 3. Quartz
column:
SCANNING
A = separating
ISOELECTROFOCUSING
column
(inner
415
tube) ; B = cooling jacket
(middle tube) ; and C = vacuum chamber (outer tube). caused by the hydrostatic pressure of the column is negligible, making it unnecessary to maintain hydrostatic equilibrium. One end of each plunger shaft (threaded) is screwed to the extension piece of the coIumn, and the other end is drilled and bears the Cheminert fittings with Teflon tubing for filling and emptying the column. The plunger shaft is inserted in the clamping collar and fastened with
FIQ. 4. Electrolyte reservoirs. Upper: A = top view of upper electrolyte reservoir; B = lower electrolyte reservoir; and C = extension pieces (top and side view) attached to the quartz column which screw directly into the threaded shaft of the electrolyte reservoirs. Middle: A = Cheminert fittings for connecting Teflon tubing to the plunger shaft; B = electrolyte reservoir without plunger shaft; C = plunger shaft of lower reservoir (note drilled holes). Lower: A = locking screw; B = Cheminert fittings for connecting auxiliary reservoir through Teflon tubing; C = electrode terminal connected to platinum wire inside the reservoir; and D = drilled holes that are filled with Styrofoam and allow the current to pass from the reservoirs to the column through a membrane that covers the outer surface of the plunger shaft.
the locking screw. Teflon tape is wrapped around the shaft at the place of attachment to the collar to ensure leak-free operation of the reservoir. The collar itself forms the electrolyte reservoir bearing a cable terminal connector and a platinum wire electrode. An auxiliary filling reservoir can be connected, when necessary, to the original reservoir by way of a Cheminert fitting and Teflon tubing. The auxiliary reservoir can prevent the original reservoir from “drying out” during overnight runs. The column and electrolyte reservoirs assembled can be seen in the photograph shown in Fig. 5.
ANALYTICAL
SCANNIKG
ISOELECTROFOCUSING
417
FIG. 5. Photograph showing details of assembled column and upper electrolyte reservoir in scanning position. A directional support for the column is visible in the upper part of the photograph.
The Column Guide The column guide provides directional guidance of the column movement perpendicularly to the light beam, a slit, air circulation for dustfree scanning environment,, and avoidance of extraneous light. A photograph of the assembled device is shown in Fig. 6. The guide was constructed from two IWO pump heads painted black, and appropriately drilled at their front to allow the light beam to pass through. The two pump heads form a chamber which is kept dust-free by circulating dry air through tubing adapters (Cole-Parmer) screwed directly into the existing threading of the heads. Extraneous light is avoided by gluing two split-ring collars lined with felt on both sides of the pump heads. The
418
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CATSIMPOOLAS
FIG. 6. Column guide. The direction of column movement is perpendicular to the plane of the paper. The direction of the light beam is from right to left of the picture (see text for details).
felt rings allow the column to slide snugly and smoothly through the guide. When the Gilford spectrophotometer was used as a detection device, two adapters from the Gilford linear transport system were attached directly to the column guide and allowed the guide to be mounted on the Gilford adapter (No. 2411), which holds the Gilford photometer and Beckman monochromator. However, the hole of the slit-holding adapter has to be opened to at, least a 15 mm diameter in order to be able to accommodate a longer slit than the one provided by Gilford with the linear transport system, or a similar piece can be constructed from wood or plastic material. A slit approximately 0.5 X 15 mm was used in these experiments. The hole in the Gilford slit-holding adapter is shown in the photograph of Fig. 6 in its original dimensions. The Scanning Device
The scanning linear
transport
device is constructed from components of the Gilford system appropriately modified to provide a more exten-
ANALYTICAL
SCANNING
ISOELECTROFOCUSING
419
FIG. 7. Scanning device : A = rotating leadscrew; B = synchronous motors; C = microswitch connected to buzzer; D = scanning indicator, which is attached to lower reservoir of the column as shown in Fig. 3; E =supporting blades; and F = motor control box.
sive linear movement (Fig. 7). The 8 rpm synchronous motor was replaced with a 40 rpm motor for faster scanning. The electrical connections inside the control box (F) remained the same. However, the knob position marked “scan rate, 2 cm/min” actually produces a scan rate of approximately 10 cm/min because of the motor exchange. The buzzer switch (C) signals the end of the scan. A long leadscrew, 18” (A), and two steel supports (E) were especially constructed. Rotation of the leadscrew (A) by the synchronous motors (B) causesthe scan indicator (D) to move upward. An extension arm screwed on the scan indicator piece supports and moves the column by being attached on the lower electrolyte reservoir (Fig. 2). The scanning device, although operative, has not been entirely troublefree because of the unbalanced weight of the column. Recently, a modified scanning device positioned directly underneath the column performed successfully under heavy use.
420
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CATSIMPOOLAS
The Electra-optical
System
This system consists of a monochromatic or filtered light source, a photomultiplier, a photometer, a strip-chart recorder, and, optionally, an integrator. An X-Y recorder can be used if the rotation of the shaft of the leadscrew is used to drive the recorder through the combination of a gear reducer and a potentiometer. Alternatively, a system offered by Esterline Angus may be preferable. The rotary shaft motion is converted by a transducer to pulses that are used as an input of the “pulse input proportional (PIP) chart drive” to advance the recorder. In this laboratory, the AISA was operated with two different systems: 1. One was based on the Gilford 2000 recording spectrophotometer equipped with a Beckman monochromator and a deuterium lamp source. After preliminary experiments with the monochromator in its normal position (as shown in Fig. 5), it was realized that the system was not scanning satisfactorily. The monochromator had to be turned “on its side” so that the light beam is perpendicular to the movement of the column, as suggested previously (2). 2. Demands for the use of the Gilford spectrophotometer for other projects in the laboratory prompted the dedication of a simpler electrooptical system to the AISA. This system consists of an inexpensive mercury-argon lamp equipped with a short-wave ultraviolet filter (Oriel), a fluorescent rod (LKB), a 280 am interference filter (LKB) , and a 17 mA AC power supply (Oriel Optics). The light system is mounted on the panel of the AISA main console with the beam directed perpendicularly to the direction of the column movement. The light beam is focused by the lens of the Gilford No. 2411 adapter. A black glass filter (LKB NO. 8025-02) was installed between the column guide and the photomultiplier. The electronics of the system-which include the photomultiplier, logarithmic converter, and integrator-were supplied by a Canalco model F microdensitometer. This can be accomplished simply by using two extension coaxial cables to get the photomultiplier out of the microdensitometer box. The optical and scanning systems of the microdensitometer are ignored. A Texas Instrument Servo-Riter II recorder supplied with the microdensitometer was used to record the analog and integrator signals. Although inexpensive lamps and filters can be employed, it should be realized that only monochromatic light of a specified wavelength (such as 280 nm) can be used for accurate quantitation of protein zones. The Temperature
Control
Unit
Temperature control is achieved by circulating middle tube of the column. A Haake thermostatic
water through the circulator, equipped
ANALYTICAL
SCANNING
ISOELECTROFOCUSING
421
with a PSC KR-30 cooler, and a circulating pump (Cole-Parmer) were used with the AISA. Care should be exercised in keeping the water clean, since the water jacket is in the path of the light beam. Helpful suggestions in this respect have been made by Hjerten (15). The exact temperature of the circulating water in the column is recorded with a YSI telethermometer. A Neslab TP-2 programmer provides controlled temperature changes in the column. This may be useful in thermal denaturation experiments studied by isoelect’rofocusing. The pH Recording
Unit
This consists of a stainless-steel box with hollow walls snuggly fitting the capillary pH electrode assembly (Beckman). Temperature control is maintained by circulating water with the unit described above through the hollow walls of the box. The electrodes were connected to a pH meter (Radiometer or Beckman). The pH meter was then connected to the Texas Instrument. Servo-Riter II recorder. A potentiometer was used to adjust the span of the recorder. A valve (Fig. 1) was used to switch the flow either of the standardization buffer or of the column effluent through the pH cell. Dense sucrose solution and a metering microburet were employed to push the liquid in the separating column upward through the pH cell. To this day, the system has not produced reliable pH records due to excessive disturbances although certain precautions in continuous pH recordings as outlined by Jonsson et al. (16) were taken into consideration. We have concluded that the use of marker proteins or peptides for calibration of the pH gradient as performed previously (1) may be the best alternative to direct pH recording. ISOELECTROFOCUSING Reagents Dense Electrolyte. This solution occupies the lower end of the separating column, and also fills the lower electrolyte reservoir (anode). It consists of 2.5 ml concentrated phosphoric acid and 25 gm sucrose made up to 50 ml with water. Light Electrolyte. This solution occupies the upper end of the separating column, and also fills the upper electrolyte reservoir (cathode). It consists of 2.5 ml ethylenediamine made to 50 ml with water. Dense Ampholyte Solution. This consists of 6 gm sucrose and 0.5 ml 40% stock solution of ampholytc (LKB) of the selected pH range made up to 10 ml with water, and is used to form the sucrose gradient. Light Ampholyte Solution. This consists of 0.5 ml 40% stock solution of ampholyte of the selected pH range, and is made up to 10 ml with
422
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CATSIMPOOLAS
water. It is also used to form the sucrose gradient. However, if a sample injection device is not available (see below), the protein sample or biological fluid is dissolved directly in this solution. Of course, by following this procedure considerable amount of the sample is wasted, and it is not placed in the middle of the sucrose gradient. Blue Dextran Solution. A stock solution of blue dextran (Pharmacia) in water of the desirable color intensity (not critical) is prepared. This solution is added to the dense electrolyte solution to color it blue, this serving as an indicator of the separation line between the dense electrolyte solution and the density gradient-ampholyte mixture. The blue dextran solution does not interfere with the isoelectrofocusing procedure. Filling
the Column
Manual filling of the column from the top as described previously (2) is laborious and awkward. According to this method the dense electrolyte is deposited first with a syringe bearing a long needle with a Teflon tubing attached to its end. The valve is opened at the bottom of the column and the void space is filled with dense electrolyte. Subsequently, preformed sucrose density gradient-ampholyte fractions (2) are layered one on top of the other by means of the Teflon tubing. The sample is placed in the middle of the sucrose gradient and finally the column is filled with the light electrolyte. However, because of the length of the column the method is slow and painstaking. An alternative method has been successfully employed. The column is filled from the bottom through a three-way valve (Hamilton). The sucrose gradient containing the ampholyte is prepared with the apparatus described by Kidby (17). The mixing chamber contains the light ampholyte solution and the sample. The dense ampholyte solution is injected into the mixing chamber with a syringe microburet (MicroMetric Instrument Co.). This allows accurate control of the volume of the ampholytesample-gradient mixture that enters the column. Usually 1.2 to 1.5 ml (actual volume, excluding dead space) was injected into the column. After injection of the gradient, the valve is switched to a syringe containing the dense electrolyte, which is injected either to a predetermined volume or until it reaches a certain mark on the column (blue dextran is useful in this respect). The valve is then closed, and the light electrolyte solution is added easily from the top with a syringe bearing a long needle. Recently a semiautomatic device that fills all the solutions in correct values from the bottom of the column, provides for sample injection in microliter volumes at a desirable location in the gradient, washes itself and the column at the end of the run, and recycles, has been designed
ANALYTICAL
SCANNING
423
ISOELECTROFOCUSING
and constructed. Details of construction are reported separately (19).
and performance
of this device
Scanning
After the column is filled, the electrolyte reservoirs are filled with the corresponding electrolytes (anode at the bottom). The current is turned on with simultaneous actuation of the elapsed time indicator. Isoelectrofocusing is performed originally at 200 V (for approximately 30 min) and then at 300V for the duration of the run. The column is scanned at selected intervals until equilibrium of focusing and diffusion is achieved. The current is not interrupted until the end of the run. If desirable, the contents of the column can be monitored with an additional detector by elution from the top, and microfractions can be collected.
0
0.0.
0.4
I
I
L 0
,_
t
INT.
$1
I 2
I 4
I
I 6
I
I 8
I
, 10
I
, 12
CM FIG. 8. Analytical scanning isoelectrofocusing in sucrose gradient of rabbit antibovine serum albumin (10 al) in pH 3-10 ampholyte range. Time of focusing 257 min; final current 0.19 mA at 500 V; temperature 20”. Scale in cm represents chart distance. The integrator signal (INT.) is included. Possible assignment of zone areas: A, gamma globulins; B, transferrins; C, albumin; D, prealbumin and haptoglobins (see text). The scan was obtained with the inexpensive argon-mercury lamp.
424
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CATSIMPOOLAS
Application
A typical analytical scanning isoelectrofocusing pattern of rabbit serum (anti-bovine albumin) in the pH 3 to 10 range is shown in Fig. 8. Possible assignment of focused zone areas was made according to the position of individual serum proteins in gel isoelectrofocusing. This pattern was included to show how the method works with a complex mixture of proteins in a biological fluid. A kinetic scanning isoelectrofocusing pattern of horse heart myoglobin (Pentex) is shown in Fig. 9. This demonstrates the advantages of the scanning method in establishing equilibrium condition between mass tranpsort and back diffusion. Additional information on the application of the method to the demonstration of multiple forms of protein subunits in dissociating media will be reported (20). It should be mentioned that the scanning method may be applicable to the separation of oligopeptides because of lack of diffusion and the advantage of wavelength choice.
Qb”‘i’
pg)
FM. 9. Kinetic scanning in pH 3-10 ampholyte
isoelectrofocusing pattern range at 300 V (16”).
““““”
of horse
B
heart
(1 l2
CM
myoglobin
(200
ANALYTICAL
SCANNING
ISOELECTROFOCUSING
425
DISCUSSION
General Comments. The basic design and construction of an in situ scanner for analytical isoelectrofocusing experiments has been described. The assembly of the AISA may be performed largely from commercially available components and parts with the aid of a small machine shop. Although this apparatus has been operated mainly with sucrose density gradients, the use of polyacrylamide gel (1) and probably Sephadex gel (13) appears to be feasible. In addition it is possible that the AISA will be useful in other electrophoretic and analytical gel filtration experiments. As a matter of fact, the apparatus was originally designed with this goal in mind, and especially for the determination of molecular weights of polypeptide chains in 6 M guanidine hydrochloride (18). The AISA can be definitely improved by incorporating a ratio recording system to cancel baseline noise and allowing the recorder to move by a proportional input pulse (PIP) chart drive mechanism. Multiple Sample Analysis. In its present form, the AISA is mainly a research instrument capable of electrofocusing no more than two samples per day. However, the importance of multiple-sample analysis especially for routine clinical applications cannot be overlooked. Quantitative isoelectrofocusing analysis of serum proteins, hemoglobins, and lipoproteins may be of value in clinical diagnostic procedures. The variety of available ampholytes offers an opportunity for high-resolution analysis of proteins of biological fluids at different pH ranges. In view of the above considerations, the construction of a multiple analytical isoelectrofocusing scanning apparatus (MAISA) appears to be desirable. This instrument may utilize advantages of programmed sequential flow of electrolytes, density gradient, and sample through multiple quartz columns which on a continuous rotational basis will be automatically filled, electrofocused, scanned, cleaned, and recycled. The instrument, after an initial 3 hr lag, may be capable of analyzing 12 samples per hour on a continuous basis, unattended. SUMMARY
An in situ scanning apparatus for analytical isoelectrofocusing experiments has been designed and constructed. The apparatus is capable of direct optical scanning of protein zones separated by isoelectric focusing in a quartz column. Sequential scans can be obtained in the presence of electrical current until equilibrium of electric mass transport and diffusion has been reached. Temperature control has been provided. Flow
426
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CATSIMPOOLAS
of reagents and sample through the column facilitates the filling procedure, prior to isoelectrofocusing, and provides the basis for subsequent future automation. REFERENCES N., AND WANG, J., AnaE. B&hem. 39, 141 (19’71). N., Sep. Sci. 6, 435 (1971). E. E., AND ACKERS, G. K., J. Biol. Chem. 243, 6315 (1968). in “Methods of Biochemical Analysis” (D. Glick, ed.), Vol. 18, p. 55. Interscience, New York, 1970. OLIVERA, B. M., BAINE, P., AND DAVIDSON, N., Biopolymers 2, 245 (1964). HOCHSTRASSER, H., LERNER, H., AND SKEGGS, JR., L. T., in “Electrophoresis” (M. Bier, ed.), Vol. 2, p. 473. Academic Press, New York, 1967. RESSLER, N., in “Electrophoresis” (M. Bier, ed.), Vol. 2, p. 493. Academic Press, New York, 1967. LOENING, U. E., Biochem. J. 102, 251 (1967). GRESSEL, J., AND WOLOWELSKY, J., Anal. Biochem. 24, 157 (1968). DRAVID, A. R., FRED~N, H., AND LARSON, S., J. Chromator. 41, 53 (1969). BORRIS, D. P., AND ARONSON, J. N., Anal. Biochem. 32, 273 (1969). WATKIN, J. E., AND MILLER, R. A., Anal. Biochem. 34, 424 (1970). FAWCETT, J. S., in “Protides of the Biological Fluids” (H. Peeters, ed.), Vol. 17, p. 409. Pergamon Press, Elmsford, N. Y., 1970. BRAKKE, M. K., ALLINGTON, R. W., AND LANGILLE, F. A., Anal. Biochem. 25, 30 (1968). HJERT~~N, S., Chromutogr. Rev. 9, 122 (1967). JONSSON. M., PETTERSON, E., AND RILBE, H., Acta Chem. &and. 23, 1553 (1969). KIDBY, i. K.; Anal. Biochem. 34, 478 (1970). FISH, W. W., MANN, K. G.. AND TANFORD, C., J. Biol. Chem. 244, 4939 (1969). CATSIMPOOLAS, N.. Anal. Rio&em. 44, 427 (1971). CATSIMPOOLAS, N., AND WANG, J., Anal. Biochem. 44, 436 (1971).
1. CATSIMPOOLAS, 2. CATSIMPOOLAS, 3. BRUMBAUGH, 4. HJERT~N, S.,
5. 6. 7. 8.
9. 10. 11. 12.
13. 14. 15. 16. 17. 18. 19. 20.
BIOCHEMISTRY
‘%‘i,
Analytical
411426
Scanning
3. Design and Operation
NICHOLAS Laboratory
(1971)
of Protein
Isoelectrofocusing:
of an in Situ Scanning
Apparatus1
CATSIMPOOLAS
Chemistry, Central Soya Research Center, Chicago, Illinois 60639
Received February
24, 1971
The advantage of in situ direct optical scanning techniques in analytical isoelectrofocusing, have been demonstrated both in polyacrylamide gel (1) and sucrose density gradient (2) media. Optical scanning methods have also been used successfully in other analytical separation techniques such as gel chromatography (3), free zone electrophoresis (4)) and density gradient, or semifluid film electrophoresis (5-7). In \situ scanning procedures should be distinguished from other optical scanning methods developed for polyacrylamide gel electrophoresis (8-12) or isoelectrofocusing (13). The latter employ scanning after termination of the experiment and in the absence of current. This imposes imitations in obtaining kinetic data and does not compensate for zone diffusion. A density gradient zone electrophoresis apparatus (14) designed for measuring electrophoretic mobilities incorporates some desirable features that have been adopted in this work but again is not truly “in situ scanning” because the current is interrupted for measurements, and the gradient column is pumped through an ultraviolet flow cell. In view of the suitability and usefulness of the in situ scanning techniques in isoelectrofocusing, electrophoresis, and gel filtration experiments at the analytical level, the development of a scanning apparatus capable of performing these procedures interchangeably was considered desirable. It is the purpose of the present paper to report the basic design of such an apparatus, and, at present, describe its operation in isoelectrofocusing experiments. DESIGN
OF THE
APPARATUS
Overall Description
A schematic diagram of the analytical isoelectrofocusing scanning apparatus (AISA) is shown in Fig. 1. The quartz isoelectrofocusing col’ References 1 and 2 are Parts 1 and 2, respectively, 411
of t&i series.
412
NICHOLAS
CATSIMPOOLAS
Sb
FIG. 1. Schematic diagram of analytical isoelect.rofocusing apparatus (AISA) : Am = ammeter; Cg = column guide ; Cl = cooler; Co = column ; Cp = capillary pH electrode assembly; Cr = circulator (thermostated) ; Eps = electrophoresis power supply; Fd = filling device ; Int = integrator; L = lamp ; Lps = lamp power supply; Ls = leadscrew ; M = monoehromator ; MC = motor control box; Mp = metering pump (or autoburet) ; P = photomultiplier; pH = pH meter; Pm = pump; PO = potentiometer; Pps = photomultiplier power supply; Pt = photometer ; Rc = recorder ; Rs = reservoir ; Sb = standardization buffer; Sm = synchronous motor ; SW = switch; Thp = thermistor probe chamber; Tm = elapsed time indicator; Tp = temperature programmer; Tth = telethermometer; Vc = vacuum pump; Vl = valve; Ws = waste.
(Co) is moved vertically by a synchronous motor (Sm) attached to the leadscrew (Ls). The movement of the motor is controlled by the motor control box (MC). The photomultiplier (P), connected to the photometer (Pt), recorder (Rc), and integrator (Int), detects the absorbance of filtered (M) light from the lamp (L) by the light-absorbing zones of materials in the separating column. The column moves through the column guide (Cg), which bears a slit positioned perpendicularly to the direction of the column movement. The electrophoresis power supply
umn
ANALYTICAL
SCANNING
ISOELECTHOFOCUSISG
413
(Eps) is connected to the electrolyte reservoirs (Rs) . The current is turned on by switches (SW) and is measured by the milli- or microammeters (Am). The duration of isoelectrofocusing is indicated by the elapsed time indicator (Tm). Temperature control for the column is provided by the thermostated water circulator (Cr). For temperatures below ambient, the cooler (Cl) circulates cooling fluid through the coils of the circulator by means of the pump (I’m). Controlled temperature changes can be obtained with the temperature programmer (Tp). The outer jacket of the column is evacuated with the vacuum pump (Vc) in order to avoid condensation of vapors at low cooling temperatures. The temperature of the circulating water is measured in a thermistor probe chamber (Thp) with a telethermometer (Tth). The pH of the column effluent is determined by passing through the capillary pH electrode assembly (Cp) connected to the recorder (Rc). A valve (Vl) is used for introduction of standardization buffer (Sb) into the pH cell. The column is filled from the bottom through a valve (Vl). A photograph of the AISA is shown in Fig. 2. The Column
and Electrolyte
Reservoirs
The Column. Suprasil T-20 (Amersil) material was used for construction of the column which consists of three concentric tubes-inner, middle, and outer corresponding to the separation compartment, cooling jacket, and vacuum jacket (Fig. 3). The outer diameters of these tubes are 4, 9, and 15 mm, and the inner diameters are 3, 7, and 13 mm, respectively. The length of the inner column is approximately 413 mm, with the middle and outer tubes relatively shorter in order to accommodate the water and vacuum outlets as shown in Fig. 4. The ends of the inner tube are connected to the electrolyte reservoirs. The Electrolyte Reservoirs. These were constructed from LKB column adapters (extensive piece, plunger shaft, and clamping collar) cut to appropriate length, and Chromatromix Cheminert fittings (Fig. 4). The extension pieces are glued to the ends of the inner tube of the column and serve as extensions of the column and as a means of screwing the column into t.he plunger shafts. The latter serve as holders of the membrane that separates the electrolyte in the electrolyte reservoir from the electrolyte inside the tube of the shaft. This is accomplished by drilling four holes (in the shape of a cross) perpendicularly to the length of the shaft connecting the outer cylindrical surface of the shaft with the inner tube. The holes are filled with a piece of Styrofoam-cut to the dimensions of the hole-ant1 covered from the outer area with two or three folds of dialysis membrane sheet (Cole-Parmer). The ~~mrhralle is held on the
NICHOLAS
CATSIMPOOLAS
Fro. 2. Photograph of different parts of the AISA: A = telethermometer; B = pH meter; C = reference pH electrode; D = capillary pH electrode assembly in hollow steel box; E = thermistor probe chamber; F = milli- and microammeters (three ranges) ; G = elapsed time indicator ; H = column with upper electrolyte reservoir; I = electrode terminal ; J = three-way valve ; K = electrophoresis power supply; L = digital absorbancemeter (for zeroing baseline) ; M = recorder; N = photometer ; 0 = scanning device ; P = motor control box ; Q = thermostated circulator; R = cooler; S = autoburet ; and T = vacuum pump.
shaft either with 2 O-rings or electrical tape and allows current to pass from the electrolyte in the reservoir to the electrolyte in the tube. As mentioned by Brakke et al. (14) in the description of their density gradient electrophoresis apparatus, the flow of liquid through these membranes
ANALYTICAL
FIG. 3. Quartz
column:
SCANNING
A = separating
ISOELECTROFOCUSING
column
(inner
415
tube) ; B = cooling jacket
(middle tube) ; and C = vacuum chamber (outer tube). caused by the hydrostatic pressure of the column is negligible, making it unnecessary to maintain hydrostatic equilibrium. One end of each plunger shaft (threaded) is screwed to the extension piece of the coIumn, and the other end is drilled and bears the Cheminert fittings with Teflon tubing for filling and emptying the column. The plunger shaft is inserted in the clamping collar and fastened with
FIQ. 4. Electrolyte reservoirs. Upper: A = top view of upper electrolyte reservoir; B = lower electrolyte reservoir; and C = extension pieces (top and side view) attached to the quartz column which screw directly into the threaded shaft of the electrolyte reservoirs. Middle: A = Cheminert fittings for connecting Teflon tubing to the plunger shaft; B = electrolyte reservoir without plunger shaft; C = plunger shaft of lower reservoir (note drilled holes). Lower: A = locking screw; B = Cheminert fittings for connecting auxiliary reservoir through Teflon tubing; C = electrode terminal connected to platinum wire inside the reservoir; and D = drilled holes that are filled with Styrofoam and allow the current to pass from the reservoirs to the column through a membrane that covers the outer surface of the plunger shaft.
the locking screw. Teflon tape is wrapped around the shaft at the place of attachment to the collar to ensure leak-free operation of the reservoir. The collar itself forms the electrolyte reservoir bearing a cable terminal connector and a platinum wire electrode. An auxiliary filling reservoir can be connected, when necessary, to the original reservoir by way of a Cheminert fitting and Teflon tubing. The auxiliary reservoir can prevent the original reservoir from “drying out” during overnight runs. The column and electrolyte reservoirs assembled can be seen in the photograph shown in Fig. 5.
ANALYTICAL
SCANNIKG
ISOELECTROFOCUSING
417
FIG. 5. Photograph showing details of assembled column and upper electrolyte reservoir in scanning position. A directional support for the column is visible in the upper part of the photograph.
The Column Guide The column guide provides directional guidance of the column movement perpendicularly to the light beam, a slit, air circulation for dustfree scanning environment,, and avoidance of extraneous light. A photograph of the assembled device is shown in Fig. 6. The guide was constructed from two IWO pump heads painted black, and appropriately drilled at their front to allow the light beam to pass through. The two pump heads form a chamber which is kept dust-free by circulating dry air through tubing adapters (Cole-Parmer) screwed directly into the existing threading of the heads. Extraneous light is avoided by gluing two split-ring collars lined with felt on both sides of the pump heads. The
418
NICHOLAS
CATSIMPOOLAS
FIG. 6. Column guide. The direction of column movement is perpendicular to the plane of the paper. The direction of the light beam is from right to left of the picture (see text for details).
felt rings allow the column to slide snugly and smoothly through the guide. When the Gilford spectrophotometer was used as a detection device, two adapters from the Gilford linear transport system were attached directly to the column guide and allowed the guide to be mounted on the Gilford adapter (No. 2411), which holds the Gilford photometer and Beckman monochromator. However, the hole of the slit-holding adapter has to be opened to at, least a 15 mm diameter in order to be able to accommodate a longer slit than the one provided by Gilford with the linear transport system, or a similar piece can be constructed from wood or plastic material. A slit approximately 0.5 X 15 mm was used in these experiments. The hole in the Gilford slit-holding adapter is shown in the photograph of Fig. 6 in its original dimensions. The Scanning Device
The scanning linear
transport
device is constructed from components of the Gilford system appropriately modified to provide a more exten-
ANALYTICAL
SCANNING
ISOELECTROFOCUSING
419
FIG. 7. Scanning device : A = rotating leadscrew; B = synchronous motors; C = microswitch connected to buzzer; D = scanning indicator, which is attached to lower reservoir of the column as shown in Fig. 3; E =supporting blades; and F = motor control box.
sive linear movement (Fig. 7). The 8 rpm synchronous motor was replaced with a 40 rpm motor for faster scanning. The electrical connections inside the control box (F) remained the same. However, the knob position marked “scan rate, 2 cm/min” actually produces a scan rate of approximately 10 cm/min because of the motor exchange. The buzzer switch (C) signals the end of the scan. A long leadscrew, 18” (A), and two steel supports (E) were especially constructed. Rotation of the leadscrew (A) by the synchronous motors (B) causesthe scan indicator (D) to move upward. An extension arm screwed on the scan indicator piece supports and moves the column by being attached on the lower electrolyte reservoir (Fig. 2). The scanning device, although operative, has not been entirely troublefree because of the unbalanced weight of the column. Recently, a modified scanning device positioned directly underneath the column performed successfully under heavy use.
420
NICHOLAS
CATSIMPOOLAS
The Electra-optical
System
This system consists of a monochromatic or filtered light source, a photomultiplier, a photometer, a strip-chart recorder, and, optionally, an integrator. An X-Y recorder can be used if the rotation of the shaft of the leadscrew is used to drive the recorder through the combination of a gear reducer and a potentiometer. Alternatively, a system offered by Esterline Angus may be preferable. The rotary shaft motion is converted by a transducer to pulses that are used as an input of the “pulse input proportional (PIP) chart drive” to advance the recorder. In this laboratory, the AISA was operated with two different systems: 1. One was based on the Gilford 2000 recording spectrophotometer equipped with a Beckman monochromator and a deuterium lamp source. After preliminary experiments with the monochromator in its normal position (as shown in Fig. 5), it was realized that the system was not scanning satisfactorily. The monochromator had to be turned “on its side” so that the light beam is perpendicular to the movement of the column, as suggested previously (2). 2. Demands for the use of the Gilford spectrophotometer for other projects in the laboratory prompted the dedication of a simpler electrooptical system to the AISA. This system consists of an inexpensive mercury-argon lamp equipped with a short-wave ultraviolet filter (Oriel), a fluorescent rod (LKB), a 280 am interference filter (LKB) , and a 17 mA AC power supply (Oriel Optics). The light system is mounted on the panel of the AISA main console with the beam directed perpendicularly to the direction of the column movement. The light beam is focused by the lens of the Gilford No. 2411 adapter. A black glass filter (LKB NO. 8025-02) was installed between the column guide and the photomultiplier. The electronics of the system-which include the photomultiplier, logarithmic converter, and integrator-were supplied by a Canalco model F microdensitometer. This can be accomplished simply by using two extension coaxial cables to get the photomultiplier out of the microdensitometer box. The optical and scanning systems of the microdensitometer are ignored. A Texas Instrument Servo-Riter II recorder supplied with the microdensitometer was used to record the analog and integrator signals. Although inexpensive lamps and filters can be employed, it should be realized that only monochromatic light of a specified wavelength (such as 280 nm) can be used for accurate quantitation of protein zones. The Temperature
Control
Unit
Temperature control is achieved by circulating middle tube of the column. A Haake thermostatic
water through the circulator, equipped
ANALYTICAL
SCANNING
ISOELECTROFOCUSING
421
with a PSC KR-30 cooler, and a circulating pump (Cole-Parmer) were used with the AISA. Care should be exercised in keeping the water clean, since the water jacket is in the path of the light beam. Helpful suggestions in this respect have been made by Hjerten (15). The exact temperature of the circulating water in the column is recorded with a YSI telethermometer. A Neslab TP-2 programmer provides controlled temperature changes in the column. This may be useful in thermal denaturation experiments studied by isoelect’rofocusing. The pH Recording
Unit
This consists of a stainless-steel box with hollow walls snuggly fitting the capillary pH electrode assembly (Beckman). Temperature control is maintained by circulating water with the unit described above through the hollow walls of the box. The electrodes were connected to a pH meter (Radiometer or Beckman). The pH meter was then connected to the Texas Instrument. Servo-Riter II recorder. A potentiometer was used to adjust the span of the recorder. A valve (Fig. 1) was used to switch the flow either of the standardization buffer or of the column effluent through the pH cell. Dense sucrose solution and a metering microburet were employed to push the liquid in the separating column upward through the pH cell. To this day, the system has not produced reliable pH records due to excessive disturbances although certain precautions in continuous pH recordings as outlined by Jonsson et al. (16) were taken into consideration. We have concluded that the use of marker proteins or peptides for calibration of the pH gradient as performed previously (1) may be the best alternative to direct pH recording. ISOELECTROFOCUSING Reagents Dense Electrolyte. This solution occupies the lower end of the separating column, and also fills the lower electrolyte reservoir (anode). It consists of 2.5 ml concentrated phosphoric acid and 25 gm sucrose made up to 50 ml with water. Light Electrolyte. This solution occupies the upper end of the separating column, and also fills the upper electrolyte reservoir (cathode). It consists of 2.5 ml ethylenediamine made to 50 ml with water. Dense Ampholyte Solution. This consists of 6 gm sucrose and 0.5 ml 40% stock solution of ampholytc (LKB) of the selected pH range made up to 10 ml with water, and is used to form the sucrose gradient. Light Ampholyte Solution. This consists of 0.5 ml 40% stock solution of ampholyte of the selected pH range, and is made up to 10 ml with
422
NICHOLAS
CATSIMPOOLAS
water. It is also used to form the sucrose gradient. However, if a sample injection device is not available (see below), the protein sample or biological fluid is dissolved directly in this solution. Of course, by following this procedure considerable amount of the sample is wasted, and it is not placed in the middle of the sucrose gradient. Blue Dextran Solution. A stock solution of blue dextran (Pharmacia) in water of the desirable color intensity (not critical) is prepared. This solution is added to the dense electrolyte solution to color it blue, this serving as an indicator of the separation line between the dense electrolyte solution and the density gradient-ampholyte mixture. The blue dextran solution does not interfere with the isoelectrofocusing procedure. Filling
the Column
Manual filling of the column from the top as described previously (2) is laborious and awkward. According to this method the dense electrolyte is deposited first with a syringe bearing a long needle with a Teflon tubing attached to its end. The valve is opened at the bottom of the column and the void space is filled with dense electrolyte. Subsequently, preformed sucrose density gradient-ampholyte fractions (2) are layered one on top of the other by means of the Teflon tubing. The sample is placed in the middle of the sucrose gradient and finally the column is filled with the light electrolyte. However, because of the length of the column the method is slow and painstaking. An alternative method has been successfully employed. The column is filled from the bottom through a three-way valve (Hamilton). The sucrose gradient containing the ampholyte is prepared with the apparatus described by Kidby (17). The mixing chamber contains the light ampholyte solution and the sample. The dense ampholyte solution is injected into the mixing chamber with a syringe microburet (MicroMetric Instrument Co.). This allows accurate control of the volume of the ampholytesample-gradient mixture that enters the column. Usually 1.2 to 1.5 ml (actual volume, excluding dead space) was injected into the column. After injection of the gradient, the valve is switched to a syringe containing the dense electrolyte, which is injected either to a predetermined volume or until it reaches a certain mark on the column (blue dextran is useful in this respect). The valve is then closed, and the light electrolyte solution is added easily from the top with a syringe bearing a long needle. Recently a semiautomatic device that fills all the solutions in correct values from the bottom of the column, provides for sample injection in microliter volumes at a desirable location in the gradient, washes itself and the column at the end of the run, and recycles, has been designed
ANALYTICAL
SCANNING
423
ISOELECTROFOCUSING
and constructed. Details of construction are reported separately (19).
and performance
of this device
Scanning
After the column is filled, the electrolyte reservoirs are filled with the corresponding electrolytes (anode at the bottom). The current is turned on with simultaneous actuation of the elapsed time indicator. Isoelectrofocusing is performed originally at 200 V (for approximately 30 min) and then at 300V for the duration of the run. The column is scanned at selected intervals until equilibrium of focusing and diffusion is achieved. The current is not interrupted until the end of the run. If desirable, the contents of the column can be monitored with an additional detector by elution from the top, and microfractions can be collected.
0
0.0.
0.4
I
I
L 0
,_
t
INT.
$1
I 2
I 4
I
I 6
I
I 8
I
, 10
I
, 12
CM FIG. 8. Analytical scanning isoelectrofocusing in sucrose gradient of rabbit antibovine serum albumin (10 al) in pH 3-10 ampholyte range. Time of focusing 257 min; final current 0.19 mA at 500 V; temperature 20”. Scale in cm represents chart distance. The integrator signal (INT.) is included. Possible assignment of zone areas: A, gamma globulins; B, transferrins; C, albumin; D, prealbumin and haptoglobins (see text). The scan was obtained with the inexpensive argon-mercury lamp.
424
NICHOLAS
CATSIMPOOLAS
Application
A typical analytical scanning isoelectrofocusing pattern of rabbit serum (anti-bovine albumin) in the pH 3 to 10 range is shown in Fig. 8. Possible assignment of focused zone areas was made according to the position of individual serum proteins in gel isoelectrofocusing. This pattern was included to show how the method works with a complex mixture of proteins in a biological fluid. A kinetic scanning isoelectrofocusing pattern of horse heart myoglobin (Pentex) is shown in Fig. 9. This demonstrates the advantages of the scanning method in establishing equilibrium condition between mass tranpsort and back diffusion. Additional information on the application of the method to the demonstration of multiple forms of protein subunits in dissociating media will be reported (20). It should be mentioned that the scanning method may be applicable to the separation of oligopeptides because of lack of diffusion and the advantage of wavelength choice.
Qb”‘i’
pg)
FM. 9. Kinetic scanning in pH 3-10 ampholyte
isoelectrofocusing pattern range at 300 V (16”).
““““”
of horse
B
heart
(1 l2
CM
myoglobin
(200
ANALYTICAL
SCANNING
ISOELECTROFOCUSING
425
DISCUSSION
General Comments. The basic design and construction of an in situ scanner for analytical isoelectrofocusing experiments has been described. The assembly of the AISA may be performed largely from commercially available components and parts with the aid of a small machine shop. Although this apparatus has been operated mainly with sucrose density gradients, the use of polyacrylamide gel (1) and probably Sephadex gel (13) appears to be feasible. In addition it is possible that the AISA will be useful in other electrophoretic and analytical gel filtration experiments. As a matter of fact, the apparatus was originally designed with this goal in mind, and especially for the determination of molecular weights of polypeptide chains in 6 M guanidine hydrochloride (18). The AISA can be definitely improved by incorporating a ratio recording system to cancel baseline noise and allowing the recorder to move by a proportional input pulse (PIP) chart drive mechanism. Multiple Sample Analysis. In its present form, the AISA is mainly a research instrument capable of electrofocusing no more than two samples per day. However, the importance of multiple-sample analysis especially for routine clinical applications cannot be overlooked. Quantitative isoelectrofocusing analysis of serum proteins, hemoglobins, and lipoproteins may be of value in clinical diagnostic procedures. The variety of available ampholytes offers an opportunity for high-resolution analysis of proteins of biological fluids at different pH ranges. In view of the above considerations, the construction of a multiple analytical isoelectrofocusing scanning apparatus (MAISA) appears to be desirable. This instrument may utilize advantages of programmed sequential flow of electrolytes, density gradient, and sample through multiple quartz columns which on a continuous rotational basis will be automatically filled, electrofocused, scanned, cleaned, and recycled. The instrument, after an initial 3 hr lag, may be capable of analyzing 12 samples per hour on a continuous basis, unattended. SUMMARY
An in situ scanning apparatus for analytical isoelectrofocusing experiments has been designed and constructed. The apparatus is capable of direct optical scanning of protein zones separated by isoelectric focusing in a quartz column. Sequential scans can be obtained in the presence of electrical current until equilibrium of electric mass transport and diffusion has been reached. Temperature control has been provided. Flow
426
NICHOLAS
CATSIMPOOLAS
of reagents and sample through the column facilitates the filling procedure, prior to isoelectrofocusing, and provides the basis for subsequent future automation. REFERENCES N., AND WANG, J., AnaE. B&hem. 39, 141 (19’71). N., Sep. Sci. 6, 435 (1971). E. E., AND ACKERS, G. K., J. Biol. Chem. 243, 6315 (1968). in “Methods of Biochemical Analysis” (D. Glick, ed.), Vol. 18, p. 55. Interscience, New York, 1970. OLIVERA, B. M., BAINE, P., AND DAVIDSON, N., Biopolymers 2, 245 (1964). HOCHSTRASSER, H., LERNER, H., AND SKEGGS, JR., L. T., in “Electrophoresis” (M. Bier, ed.), Vol. 2, p. 473. Academic Press, New York, 1967. RESSLER, N., in “Electrophoresis” (M. Bier, ed.), Vol. 2, p. 493. Academic Press, New York, 1967. LOENING, U. E., Biochem. J. 102, 251 (1967). GRESSEL, J., AND WOLOWELSKY, J., Anal. Biochem. 24, 157 (1968). DRAVID, A. R., FRED~N, H., AND LARSON, S., J. Chromator. 41, 53 (1969). BORRIS, D. P., AND ARONSON, J. N., Anal. Biochem. 32, 273 (1969). WATKIN, J. E., AND MILLER, R. A., Anal. Biochem. 34, 424 (1970). FAWCETT, J. S., in “Protides of the Biological Fluids” (H. Peeters, ed.), Vol. 17, p. 409. Pergamon Press, Elmsford, N. Y., 1970. BRAKKE, M. K., ALLINGTON, R. W., AND LANGILLE, F. A., Anal. Biochem. 25, 30 (1968). HJERT~~N, S., Chromutogr. Rev. 9, 122 (1967). JONSSON. M., PETTERSON, E., AND RILBE, H., Acta Chem. &and. 23, 1553 (1969). KIDBY, i. K.; Anal. Biochem. 34, 478 (1970). FISH, W. W., MANN, K. G.. AND TANFORD, C., J. Biol. Chem. 244, 4939 (1969). CATSIMPOOLAS, N.. Anal. Rio&em. 44, 427 (1971). CATSIMPOOLAS, N., AND WANG, J., Anal. Biochem. 44, 436 (1971).
1. CATSIMPOOLAS, 2. CATSIMPOOLAS, 3. BRUMBAUGH, 4. HJERT~N, S.,
5. 6. 7. 8.
9. 10. 11. 12.
13. 14. 15. 16. 17. 18. 19. 20.