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Centrifugation in field-aligning capsules: Analytical centrifugation in preparative rotors
Centrifugation in Field-Aligning Capsules : Analytical Centrifugation in Preparative Rotors’ Robert Virus
Laboratory,
C. Backus University
and Robley C. Williams of
California,
Berkeley,
California
ReceivedSeptember8,1953
Field-aligning capsules (1) which are supported by flotation in conventional angle-head rotors, and are removable from the rotors subsequent to centrifugation for examination by optical methods, provide a relatively inexpensive, convenient, and versatile method for analytical ultracentrifugation. The method is particularly suitable for exploratory work requiring occasional observations on the efficacy of preparative methods directed toward isolation of biologically active materials of high molecular weight, and for the routine examination of specimens of familiar composition. The advantages of centrifugation in capillary tubes have been pointed out by Elford (2) and by McBain and others (3). Convection is minimized and lateral motion of the fluid contents is arrested in tubes with bores approximately 1 mm. or smaller in diameter. Such tubes have been removed and manipulated after centrifugation for various kinds of optical analyses (3). Although the capsular method is not limited to cells of “capillary” bore as defined above, the ability to centrifuge very small samples is frequently advantageous, and capsules of microscopic bore can be used if desirable. Centrifugation in field-aligning capsules is accomplished by flotation of glass or quartz capsulesin a liquid medium adjusted to proper density which in turn is contained within the ordinary plastic tubes of a preparative centrifuge rotor. As the rotor of the centrifuge accelerates, the capsules are constantly aligned with their long axes parallel to the existing 1 This research has been supported in large part, by a grant from the American Cancer Society upon recommendation of the Committee on Growth of the National Research Council. 434
CENTRIFUGATION
STUDIES
435
field of force because of the low center of gravity and the orienting effect of an enclosed bubble. Density gradients established by sedimentation remain normal to the walls of the capsulesand to the force field t’hroughout the centrifugation cycle. In effect, the capsules orient themselves as though they were mounted as swinging cups with universal bearings. PREPARATION
OF CAPSULES
The preparation of the capsules for analytical centrifugation differs in two respects from the procedure previously described (1). In order to minimize boundary distortions that might be caused by a meniscus of large curvature and to provide a well-defined plane reference surface for analysis, the inner wall of the capsule is rendered hydrophobic by treatment with one of the proprietary mixtures of the methyl chlorosilanes (e.g., Dri-Film 9987, General Electric Co.) prior to admission of the solution to be centrifuged. The Dri-Film may be drawn in thevapor state through the capsules when they are in the initial pipet stage [see Fig. la of Ref. (l)]. The solution for centrifugation must be introduced into the hydrophobic pipets with the aid of some external force, such as that produced by suction from a hypodermic syringe joined to the pipet with a perforated vaccine-bottle stopper. ,4 second preparative detail in the procedure for analytical centrifugation is :I thorough mixing of the encapsulated solution prior to centrifugation. When samplings are made from a bulk solution, mixing is best done before encapsulation with no further additions of solvent to the capsule. Mixing may be done after encapsulation, however, by alternately transposing the enclosed air bubble from end to end by a series of momentary centrifugations in a conical tube in a clinicatype centrifuge. Duplicate analyses may bc run on the same capsule by remixing the capsule contents and recentrifuging.
CENTRIFUGATION
An evaluation may be made of the centrifugal field existing at the surface of the solution within the capsule when the rotor is turning at known speed by determining the distance r, from the rotor axis t,o the meniscus (Fig. 1). It is to be noted that the value of 1, , the distancealong the center line of the plastic tube from the plane of its top to the intersection of center line and meniscus, is independent of the tilt of the tube or of the t’ilt of the meniscuswith respect to the tube as long as the meniscus is within the cylindrical portion of the tube. Hence, the value of II can be ascertained with the filled tube conveniently held in a vertical, or near-vertical position. The tube is inserted in the rotor and t,he distance I, obtained. If 8 is the rotor angle, then rm = (I, + Zt) sin 8. The distance from the meniscus of the flotation liquid to the surface of the solution within the capsule must be added to r, for precise calculations of centrifugal field.
436
R.
‘2.
BACKUS
AND
R.
C.
WILLIAMS
When low-speed centrifugation is performed, the tube can be left partially filled with the solution used for floating the capsule, but for fields exceeding that at which the plastic tube might collapse it is of course necessary to fill the tube completely. This can be done without appreciably disturbing the flotation equilibrium by layering mineral oil over the aqueous solution. It has been observed that boundaries resulting from centrifugally produced density differences within capsules of small diameter, 3 mm. or less, are very stable if the capsules are kept upright and are not subjected to localized temperature changes or greater shaking than that occasioned by careful handling.
FIG. 1. Section of centrifuge rotor at speed, showing self-aligning capsule in position in partially filled centrifuge tube. 1, is distance from centrifuge axis to top of tube; It is distance from top of tube to intersection of meniscus with axis of tube; r, is radial distance from rotor axis to meniscus in tube; 0 istherotor angle.
CENTRIFUGATION ANALYTICAL
437
STUDIES PRODCEDURES
Two kinds of optical analyses of the sedimentation boundaries have been used in this laboratory. One method makes use of the schlieren optics of an electrophoresis apparatus (Perkin-Elmer, model 38), and the other employs the differential absorption analysis obtainable with a spectrophotometer (Beckman, model IIU). In the first-mentioned method of analysis, the capsule is taken from the flotation medium by lowering the end of a section of glass tubing over it, withdrawing it pipetwise, rinsing, and transferring it to a jig which holds it vertically in the normal electrophoresis cell position for scanning. The water bath of the instrument is kept at the same temperature as the rotor in which the capsule leas centrifuged. Iqor scanning in the spectrophotometer, the capsule is dried with absorbent paper after rinsing with distilled water to remove the salt solution used for Aot,ation. For convenience in handling and to preclude unnecessary contact with the fingers, the capsule can be mounted on a short piece of sticky tape. The spectrophotometer is arranged for scanning through the horizontal boundaries by laying the instrument on its side to orient the exit slit parallel to the boundaries. 11 reduced astigmatic image of the exit slit is focused in the plane of the capsule 1)~ mounting across the round aperture in the face of the “cell compartment mounting block” (Beckman Bull. 91-G), a 7.mm. diameter fused silica rod w-it.h its axis parallel to the slit. The capsule can be mounted on an arm projectring from a cathct,on~eter or any other apparatus which will provide incremental fine-scale motion in a vertical direction.
Two materials of greatly differing sedimentation rates have been used in order to test the range of applicability of these methods of boundary formation and analysis. Bushy stunt virus (RN) has an accepted slow of 132 (4) and hence is large enough to he sedimented in the Servall model SS-1 centrifuge. A 0.5 y0 suspension in 0.05 M phosphate buffer, pH 7, was capsule-centrifuged for an effective 40 min. with the rotor spinning at 12,000 r.p.m. The distance from rotor axis to the meniscus in the capsule was 8.7 cm. The temperat,ure of the rotor and contents was kept constant at 21” by loading the rotor at 21” and performing the centrifugation in a room at 5”; at this room temperat’ure t(he heat generated by motor friction and motor windage was balanced by heat lost through air convection. The schlieren pattern (Fig. 2) obtained immediately after centrifugation shows t’hat the BSV boundary had moved 0.44 cm. from
th
Calculation
= 125 for the BSV material, value of 132.
from
these
in satisfactory
data
gives
a value
of
sZoW
agreement with the accept)cd
438
R.
C.
BACKUS
AND
h 0.6
R.
C.
WILLIAMS
-
I,
DISTANCE
FROM
MENISCUS
IN
mm.
FIG. 2. Sedimentation boundaries of bushy stunt virus centrifuged in fieldaligning capsule within a Servall SS-1 rotor. Left: schlieren pattern, capsule spun 40 min. at 14,100 X g, arrow indicates direction of sedimentation from meniscus, length of arrow is equivalent to 10 mm. on capsule. Right: spectrophotometric analysis at 300 rnp, capsule spun 25 min. at 14,100 X g. Exit slit 0.3 mm. wide, reduced slit image 0.09 mm. wide, is shown drawn to scale.
Three capsular centrifugations of BSV have been analyzed spectrophotometrically in order to evaluate the precision of this method for locating boundaries and determining their shapes. The result of such an analysis conducted at 300 rnp is shown at the right in Fig. 2, for the case of a 1 y0 solution of BSV in distilled water sedimented for 25 min. at 12,000 r.p.m. in the Servall model SS-1. As can be seenfrom the graph, the half-transmission breadth of the BSV boundary is only 0.22 mm., although the breadth of the reduced geometrical slit image is itself 0.09 mm. The corrected half-transmission breadth (approximately the same in this case as the half-concentration breadth) of this boundary is approximately 0.13 mm. Bovine plasma albumin, which has an sZoW value of 4.09 (5) in a concentration of 0.5 %, was chosen as a material for a test of the method of capsular contrifugation with much smaller sedimenting entities than viruses. A 0.5 y0 solution of albumin in 0.05 M phosphate buffer, pH 7, was centrifuged at 100,000 X g for 2 hr. at 25’ in a quartz capsule carried in the 40 rotor of a Spinco model L preparative ultracentrifuge. Figure 3 shows a schlieren pattern of the boundary, broadened presumably by molecular diffusion. An szo w = 4.4 is calculated for this sedimentation run.
CENTRIFUGATION
439
STUDIES
Spectrophotometric analyses can be performed at two or more wavelengths to aid in identifying the sedimenting components wit’hin capsules. For illustration, a model system was made up of a mixture of BSV and hemocyanine (Helix: aspersa), both in a concentration of approximately 0.01%. The preparation was centrifuged in a Spinco model L machine set for 20,000 r.p.m., with the 21-rotor. The instrument was preset to cycle through 40 min. (this does not include time required for coming to rest).
FIG. 3. Schlieren pattern of bovine plasma albumin centrifuged for 2 hr. at 100,000 X q in a field-aligning capsule within a Spinco L-40 rotor. Arrow indicates direction of sedimentation from meniscus, length of arrow is equivalent to 5 mm. on capsule.
I
I
DISTAIU~E
I FROM
I
+260mlr +26Omp I 1
IkNISCUS
fN” mm.
-
.
FIG. 4. Sedimentation boundaries of a mixture of bushy stunt virus and hemocyanine (Neliz aspersa) centrifuged for approximately 20 min. at 20,000 X q in field-aligning capsules within a Spinco L-21 rotor. Left: light absorption analysis. Concentration of each component was approximately O.Ol$‘& in 0.05 M phosphate buffer at pH 6.8. Right: schlieren pattern of the samr mixture with both sedimenting components in 50-fold greater concentration than that used for spectrophotometric analysis. Arrow indicates direction of sedimentation from meniscus! length of arrow is equivalent to 10 mm. on capsule.
440
R.
C.
BACKUS
AND
R.
C.
WILLIAMS
The boundaries in the capsule were photometrically scanned at two wavelengths, 260 and 280 rnp, with the result as shown in Fig. 4. The component which had sedimented the more rapidly during centrifugation has significantly greater light absorption at 260 rnp than at 280 mp. The curves identify correctly the faster-moving component with BSV, since the virus is known to contain about 17 % nucleic acid, whereas hemocyanine contains none. A schlieren pattern of the same mixture at a concentration 50-fold greater than that employed for the spectrophotometric analysis is shown at the right in Fig. 4. DISCUSSION
Capsular analytical ultracentrifugation has an immediate attractiveness in its simplicity and inexpensiveness compared with the much more elaborate conventional machines designed primarily for analytical use. By the method here described fairly precise sedimentation values can be determined for entities with an s20 g 50 with apparatus no more elaborate than a Servall SS-1 centrifuge or its equivalent and some means for locating the boundaries. For the slower-sedimenting particles a higher-speed centrifuge such as the Spinco model L machine is required. It appears that the vibrations encountered in the operation of a Servall centrifuge have no deleterious effect upon boundary sharpness, nor does a forced stopping of the rotor by hand, in a time as short as 50 sec., from 12,000 r.p.m. The use of capsules of 3-6 mm. diameter permits sampling to be done on the capsule contents for biological or chemical assay. The capsule is scored and broken off above the meniscus to allow sampling. Capillary centrifugation, somewhat analogous to the type of centrifugation just described, has been employed with the aid of swinging-cup rotors (6-8) and in fixed horizontal position in the rotor of an air-driven “spinning-top” centrifuge (2). The unique aspect of the self-aligning capsular method is the complete smoothness and universal orientation of the flotation ‘
CENTRIFUGATIO~
STUDIES
441
can be varied to suit the need of the individual investigation. When spectrophotometric analyses of the boundaries are performed at two or mure wavelengths and the solution contains several sedimenting componems of differing spectlrochemical compositions, an indicat.ion may be obtained of both the position and the nature of t’he components. Information of this kind is useful in differentiating, for example, boundaries of components containing nucleic acid from accompanying protein materials wit’h somewhat different sedimentation characteristics. The use of the spectrophotometer also permits location of the components of some materials in considerably lower concentrations than is possible with schlieren optics.
SUMMARY Details are given for the preparation, centifugation, and analysis of field-aligning capsules. A new method of combining scanning with differential absorption analysis utilizing the Beckman spectrophotometer is described for locating and identifying boundaries within capsules. Application of the methods are demonstrated using bushy stunt virus, hemocyanine (Helix aspers,a), and bovine serum albumin
REFERENCES 1. BACKUS+ &AND WILLIAMS, R. C.,Sciencell7,221 (1951). 2. ELFORD, W. J., &it. J. Expll. Pathot. 17,399 (1%). 3. MCBAIN, J. W., Colloid Science, Chap. 16. D. C. Heath Co., Boston, 1950. 4. LAUFFER, M. A., AND STANLEY, W. M., in Alexander (ed)., Colloid Chemist,ry, Vol. V. Reinhold Publ. Co., New York, 1944. 5. KEGELES, G., AND GUTTER, F. J., J, Am. C/tern. sot, 73, 3770 (1951). CL PULSUN, A., Na&re 148, 593 (1941). 7. KAHLER, H., AND LLOYDJ. J., JR.,J.P~~.& Colloid Chem. &,1344 (1951). 8. RRAKKE, M. K., BLACK, L. M,, AND WYCHOFF, R. W. G., Am. J. Botany 38, 332 (1951).
Laboratory,
C. Backus University
and Robley C. Williams of
California,
Berkeley,
California
ReceivedSeptember8,1953
Field-aligning capsules (1) which are supported by flotation in conventional angle-head rotors, and are removable from the rotors subsequent to centrifugation for examination by optical methods, provide a relatively inexpensive, convenient, and versatile method for analytical ultracentrifugation. The method is particularly suitable for exploratory work requiring occasional observations on the efficacy of preparative methods directed toward isolation of biologically active materials of high molecular weight, and for the routine examination of specimens of familiar composition. The advantages of centrifugation in capillary tubes have been pointed out by Elford (2) and by McBain and others (3). Convection is minimized and lateral motion of the fluid contents is arrested in tubes with bores approximately 1 mm. or smaller in diameter. Such tubes have been removed and manipulated after centrifugation for various kinds of optical analyses (3). Although the capsular method is not limited to cells of “capillary” bore as defined above, the ability to centrifuge very small samples is frequently advantageous, and capsules of microscopic bore can be used if desirable. Centrifugation in field-aligning capsules is accomplished by flotation of glass or quartz capsulesin a liquid medium adjusted to proper density which in turn is contained within the ordinary plastic tubes of a preparative centrifuge rotor. As the rotor of the centrifuge accelerates, the capsules are constantly aligned with their long axes parallel to the existing 1 This research has been supported in large part, by a grant from the American Cancer Society upon recommendation of the Committee on Growth of the National Research Council. 434
CENTRIFUGATION
STUDIES
435
field of force because of the low center of gravity and the orienting effect of an enclosed bubble. Density gradients established by sedimentation remain normal to the walls of the capsulesand to the force field t’hroughout the centrifugation cycle. In effect, the capsules orient themselves as though they were mounted as swinging cups with universal bearings. PREPARATION
OF CAPSULES
The preparation of the capsules for analytical centrifugation differs in two respects from the procedure previously described (1). In order to minimize boundary distortions that might be caused by a meniscus of large curvature and to provide a well-defined plane reference surface for analysis, the inner wall of the capsule is rendered hydrophobic by treatment with one of the proprietary mixtures of the methyl chlorosilanes (e.g., Dri-Film 9987, General Electric Co.) prior to admission of the solution to be centrifuged. The Dri-Film may be drawn in thevapor state through the capsules when they are in the initial pipet stage [see Fig. la of Ref. (l)]. The solution for centrifugation must be introduced into the hydrophobic pipets with the aid of some external force, such as that produced by suction from a hypodermic syringe joined to the pipet with a perforated vaccine-bottle stopper. ,4 second preparative detail in the procedure for analytical centrifugation is :I thorough mixing of the encapsulated solution prior to centrifugation. When samplings are made from a bulk solution, mixing is best done before encapsulation with no further additions of solvent to the capsule. Mixing may be done after encapsulation, however, by alternately transposing the enclosed air bubble from end to end by a series of momentary centrifugations in a conical tube in a clinicatype centrifuge. Duplicate analyses may bc run on the same capsule by remixing the capsule contents and recentrifuging.
CENTRIFUGATION
An evaluation may be made of the centrifugal field existing at the surface of the solution within the capsule when the rotor is turning at known speed by determining the distance r, from the rotor axis t,o the meniscus (Fig. 1). It is to be noted that the value of 1, , the distancealong the center line of the plastic tube from the plane of its top to the intersection of center line and meniscus, is independent of the tilt of the tube or of the t’ilt of the meniscuswith respect to the tube as long as the meniscus is within the cylindrical portion of the tube. Hence, the value of II can be ascertained with the filled tube conveniently held in a vertical, or near-vertical position. The tube is inserted in the rotor and t,he distance I, obtained. If 8 is the rotor angle, then rm = (I, + Zt) sin 8. The distance from the meniscus of the flotation liquid to the surface of the solution within the capsule must be added to r, for precise calculations of centrifugal field.
436
R.
‘2.
BACKUS
AND
R.
C.
WILLIAMS
When low-speed centrifugation is performed, the tube can be left partially filled with the solution used for floating the capsule, but for fields exceeding that at which the plastic tube might collapse it is of course necessary to fill the tube completely. This can be done without appreciably disturbing the flotation equilibrium by layering mineral oil over the aqueous solution. It has been observed that boundaries resulting from centrifugally produced density differences within capsules of small diameter, 3 mm. or less, are very stable if the capsules are kept upright and are not subjected to localized temperature changes or greater shaking than that occasioned by careful handling.
FIG. 1. Section of centrifuge rotor at speed, showing self-aligning capsule in position in partially filled centrifuge tube. 1, is distance from centrifuge axis to top of tube; It is distance from top of tube to intersection of meniscus with axis of tube; r, is radial distance from rotor axis to meniscus in tube; 0 istherotor angle.
CENTRIFUGATION ANALYTICAL
437
STUDIES PRODCEDURES
Two kinds of optical analyses of the sedimentation boundaries have been used in this laboratory. One method makes use of the schlieren optics of an electrophoresis apparatus (Perkin-Elmer, model 38), and the other employs the differential absorption analysis obtainable with a spectrophotometer (Beckman, model IIU). In the first-mentioned method of analysis, the capsule is taken from the flotation medium by lowering the end of a section of glass tubing over it, withdrawing it pipetwise, rinsing, and transferring it to a jig which holds it vertically in the normal electrophoresis cell position for scanning. The water bath of the instrument is kept at the same temperature as the rotor in which the capsule leas centrifuged. Iqor scanning in the spectrophotometer, the capsule is dried with absorbent paper after rinsing with distilled water to remove the salt solution used for Aot,ation. For convenience in handling and to preclude unnecessary contact with the fingers, the capsule can be mounted on a short piece of sticky tape. The spectrophotometer is arranged for scanning through the horizontal boundaries by laying the instrument on its side to orient the exit slit parallel to the boundaries. 11 reduced astigmatic image of the exit slit is focused in the plane of the capsule 1)~ mounting across the round aperture in the face of the “cell compartment mounting block” (Beckman Bull. 91-G), a 7.mm. diameter fused silica rod w-it.h its axis parallel to the slit. The capsule can be mounted on an arm projectring from a cathct,on~eter or any other apparatus which will provide incremental fine-scale motion in a vertical direction.
Two materials of greatly differing sedimentation rates have been used in order to test the range of applicability of these methods of boundary formation and analysis. Bushy stunt virus (RN) has an accepted slow of 132 (4) and hence is large enough to he sedimented in the Servall model SS-1 centrifuge. A 0.5 y0 suspension in 0.05 M phosphate buffer, pH 7, was capsule-centrifuged for an effective 40 min. with the rotor spinning at 12,000 r.p.m. The distance from rotor axis to the meniscus in the capsule was 8.7 cm. The temperat,ure of the rotor and contents was kept constant at 21” by loading the rotor at 21” and performing the centrifugation in a room at 5”; at this room temperat’ure t(he heat generated by motor friction and motor windage was balanced by heat lost through air convection. The schlieren pattern (Fig. 2) obtained immediately after centrifugation shows t’hat the BSV boundary had moved 0.44 cm. from
th
Calculation
= 125 for the BSV material, value of 132.
from
these
in satisfactory
data
gives
a value
of
sZoW
agreement with the accept)cd
438
R.
C.
BACKUS
AND
h 0.6
R.
C.
WILLIAMS
-
I,
DISTANCE
FROM
MENISCUS
IN
mm.
FIG. 2. Sedimentation boundaries of bushy stunt virus centrifuged in fieldaligning capsule within a Servall SS-1 rotor. Left: schlieren pattern, capsule spun 40 min. at 14,100 X g, arrow indicates direction of sedimentation from meniscus, length of arrow is equivalent to 10 mm. on capsule. Right: spectrophotometric analysis at 300 rnp, capsule spun 25 min. at 14,100 X g. Exit slit 0.3 mm. wide, reduced slit image 0.09 mm. wide, is shown drawn to scale.
Three capsular centrifugations of BSV have been analyzed spectrophotometrically in order to evaluate the precision of this method for locating boundaries and determining their shapes. The result of such an analysis conducted at 300 rnp is shown at the right in Fig. 2, for the case of a 1 y0 solution of BSV in distilled water sedimented for 25 min. at 12,000 r.p.m. in the Servall model SS-1. As can be seenfrom the graph, the half-transmission breadth of the BSV boundary is only 0.22 mm., although the breadth of the reduced geometrical slit image is itself 0.09 mm. The corrected half-transmission breadth (approximately the same in this case as the half-concentration breadth) of this boundary is approximately 0.13 mm. Bovine plasma albumin, which has an sZoW value of 4.09 (5) in a concentration of 0.5 %, was chosen as a material for a test of the method of capsular contrifugation with much smaller sedimenting entities than viruses. A 0.5 y0 solution of albumin in 0.05 M phosphate buffer, pH 7, was centrifuged at 100,000 X g for 2 hr. at 25’ in a quartz capsule carried in the 40 rotor of a Spinco model L preparative ultracentrifuge. Figure 3 shows a schlieren pattern of the boundary, broadened presumably by molecular diffusion. An szo w = 4.4 is calculated for this sedimentation run.
CENTRIFUGATION
439
STUDIES
Spectrophotometric analyses can be performed at two or more wavelengths to aid in identifying the sedimenting components wit’hin capsules. For illustration, a model system was made up of a mixture of BSV and hemocyanine (Helix: aspersa), both in a concentration of approximately 0.01%. The preparation was centrifuged in a Spinco model L machine set for 20,000 r.p.m., with the 21-rotor. The instrument was preset to cycle through 40 min. (this does not include time required for coming to rest).
FIG. 3. Schlieren pattern of bovine plasma albumin centrifuged for 2 hr. at 100,000 X q in a field-aligning capsule within a Spinco L-40 rotor. Arrow indicates direction of sedimentation from meniscus, length of arrow is equivalent to 5 mm. on capsule.
I
I
DISTAIU~E
I FROM
I
+260mlr +26Omp I 1
IkNISCUS
fN” mm.
-
.
FIG. 4. Sedimentation boundaries of a mixture of bushy stunt virus and hemocyanine (Neliz aspersa) centrifuged for approximately 20 min. at 20,000 X q in field-aligning capsules within a Spinco L-21 rotor. Left: light absorption analysis. Concentration of each component was approximately O.Ol$‘& in 0.05 M phosphate buffer at pH 6.8. Right: schlieren pattern of the samr mixture with both sedimenting components in 50-fold greater concentration than that used for spectrophotometric analysis. Arrow indicates direction of sedimentation from meniscus! length of arrow is equivalent to 10 mm. on capsule.
440
R.
C.
BACKUS
AND
R.
C.
WILLIAMS
The boundaries in the capsule were photometrically scanned at two wavelengths, 260 and 280 rnp, with the result as shown in Fig. 4. The component which had sedimented the more rapidly during centrifugation has significantly greater light absorption at 260 rnp than at 280 mp. The curves identify correctly the faster-moving component with BSV, since the virus is known to contain about 17 % nucleic acid, whereas hemocyanine contains none. A schlieren pattern of the same mixture at a concentration 50-fold greater than that employed for the spectrophotometric analysis is shown at the right in Fig. 4. DISCUSSION
Capsular analytical ultracentrifugation has an immediate attractiveness in its simplicity and inexpensiveness compared with the much more elaborate conventional machines designed primarily for analytical use. By the method here described fairly precise sedimentation values can be determined for entities with an s20 g 50 with apparatus no more elaborate than a Servall SS-1 centrifuge or its equivalent and some means for locating the boundaries. For the slower-sedimenting particles a higher-speed centrifuge such as the Spinco model L machine is required. It appears that the vibrations encountered in the operation of a Servall centrifuge have no deleterious effect upon boundary sharpness, nor does a forced stopping of the rotor by hand, in a time as short as 50 sec., from 12,000 r.p.m. The use of capsules of 3-6 mm. diameter permits sampling to be done on the capsule contents for biological or chemical assay. The capsule is scored and broken off above the meniscus to allow sampling. Capillary centrifugation, somewhat analogous to the type of centrifugation just described, has been employed with the aid of swinging-cup rotors (6-8) and in fixed horizontal position in the rotor of an air-driven “spinning-top” centrifuge (2). The unique aspect of the self-aligning capsular method is the complete smoothness and universal orientation of the flotation ‘
CENTRIFUGATIO~
STUDIES
441
can be varied to suit the need of the individual investigation. When spectrophotometric analyses of the boundaries are performed at two or mure wavelengths and the solution contains several sedimenting componems of differing spectlrochemical compositions, an indicat.ion may be obtained of both the position and the nature of t’he components. Information of this kind is useful in differentiating, for example, boundaries of components containing nucleic acid from accompanying protein materials wit’h somewhat different sedimentation characteristics. The use of the spectrophotometer also permits location of the components of some materials in considerably lower concentrations than is possible with schlieren optics.
SUMMARY Details are given for the preparation, centifugation, and analysis of field-aligning capsules. A new method of combining scanning with differential absorption analysis utilizing the Beckman spectrophotometer is described for locating and identifying boundaries within capsules. Application of the methods are demonstrated using bushy stunt virus, hemocyanine (Helix aspers,a), and bovine serum albumin
REFERENCES 1. BACKUS+ &AND WILLIAMS, R. C.,Sciencell7,221 (1951). 2. ELFORD, W. J., &it. J. Expll. Pathot. 17,399 (1%). 3. MCBAIN, J. W., Colloid Science, Chap. 16. D. C. Heath Co., Boston, 1950. 4. LAUFFER, M. A., AND STANLEY, W. M., in Alexander (ed)., Colloid Chemist,ry, Vol. V. Reinhold Publ. Co., New York, 1944. 5. KEGELES, G., AND GUTTER, F. J., J, Am. C/tern. sot, 73, 3770 (1951). CL PULSUN, A., Na&re 148, 593 (1941). 7. KAHLER, H., AND LLOYDJ. J., JR.,J.P~~.& Colloid Chem. &,1344 (1951). 8. RRAKKE, M. K., BLACK, L. M,, AND WYCHOFF, R. W. G., Am. J. Botany 38, 332 (1951).