A schlieren optics accessory for use with the preparative ultracentrifuge

ANALYTICAL

BIOCHEMISTRY

31, 218-226

(1969)

A Schlieren

Optics Accessory for Use with the Preparative Ultracentrifuge 0. M. GRIFFITH AND L. GROPPER

Be&man

Instruments,

Inc., Spinco Division, 1117 California Palo Alto, Califomzia 9.4304 Received April

Avenue,

2, 1969

Since the development of Svedberg’s instrument, ultracentrifugation has been divided, more or less, into two areas of instrumentation, namely, preparative and analytical. Preparative ultracentrifuges are commonly used to separate particles on the basis of certain physical properties, while the analytical ultracentrifuges are used to determine what those physical properties might be. The schlieren optical system, which is an integral part of the analytical ultracentrifuge, can now be used as an accessory with an analytical rotor for both direct observation and photography of boundary movements of macromolecules such as proteins in the preparative ultracentrifuge. The following experiments can be made with the accessory, when installed on the Beckman model LZ-65B or the model L preparative u1tracentrifuges.l (a) Sedimentation velocity studies for the determination of sedimentation coefficients, (b) synthetic boundary studies for determining solution concentrations and diffusion coefficients, and (c) sedimentation equilibrium studies for molecular weight determinations. In this report the experiments performed were accomplished with the optics accessory installed on the model LZ-65B ultracentrifuge. The system of the preparative ultracentrifuge and the schlieren optics accessory cannot be considered equivalent in precision to the analytical ultracentrifuge as typified by the Beckman model El. Rather it is suited for less precise work and can be used as a teaching aid for demonstrating the basic principles of the more sensitive instrument. Unlike the analytical ultracentrifuge, the schlieren optics accessory is designed only to analyze schlieren patterns from ultracentrifuge experiments. Rayleigh interference patterns cannot 1 Manufactured California.

by Spinco

Division,

Beckman 218

Instruments,

Inc., Palo

Alto,

SCHLIEREN

OPTICS

ACCESSORY

219

be observed with this accessory at present, but this may be possible in the future. GENERAL

DESCRIPTION

The schlieren optics accessory is an optical track bearing mirrors, lenses, light source, schlieren diaphragm, viewing aperture, and a Polaroid? camera, most of which are installed in a vertical column over two holes in the rotor chamber door. Two angled mirrors, as well as the collimating and condensing lenses, are in the rotor chamber itself, where they direct light downward from the light source and then upward through the rotor cell containing the sample. A separate “switch box” controls the light source. The optical

FIG. 1. Photograph of optical accessory model LZ-65B preparative ultracentrifuge. paratus are also shown. 2 Manufactured

by Polaroid

Corporation,

mounted on rotor chamber door of The “switch box” and viewing apCambridge

39, Massachusetts.

220

GRIFFITH

AND

GROPPER

track is attached to the rotor chamber door by three screws, and the mirrors and lenses in the chamber door are attached to a modified base plate of the centrifuge drive. Both the optical track and the modified base plate with mirrors and lenses can be removed to allow normal use of any preparative rotor rated for the instrument. Two screw plugs are also provided to seal the two holes in the rotor chamber door after the optical track is removed. Figure 1 shows a photograph of the optical accessory mounted on the rotor chamber door and Figure 2 is a diagram indicating the light path from the Viewi* Mirmr

Cylindrical

Cbmbw

-

Lam

Door

All.0 Rotor and Call Assembly

Frcnt Surface Mirrors

FIG. 2. Diagram indicating light path from light source to camera. Lenses, wire diaphragm, and filter are shown with respect to their positions in the accessory unit.

SCHLIEREN

OPTICS

ACCESSORY

221

light source to the camera. The only difference between the rotor and that of the model E rotor, type An-D’, is that it is modified at the base to accept the preparative instrument’s driveshaft. All standard 12 mm single- and double-sector model E rotor cells, with appropriate counterbalances, can be used; however, the sytem is not sensitive enough to use the multi “short-column equilibrium” cells, wedge windows, or wedge centerpieces. When a double-sector cell is used, the solvent sector provides a baseline for the schlieren curve generated by the sample sector. A duplicate run with solvent alone provides a baseline when a single-sector cell is used. For optimum image, a knob at the top of the optical column allows manual rotation of the schlieren diaphragm from angles 45” to 90” in 0.5” increments. Two wire diaphragms are provided. One having 0.010” thickness is used for sedimentation velocity runs, and the other having 0.003” thickness is used for sedimentation equilibrium runs. PermissibIe run speed is 60,000 rpm (maximum for the An-D rotor) or the centrifuge drive maximum speed, whichever is lower. During the run, the operator has manual control of light source and photography ; exposure time and intervals between exposures are determined and instigated by the operator. At any time during the run, the camera may be easily removed from the optical column and replaced by the viewing apparatus for direct viewing of the image. (Viewer and camera are used in the same mounting.) Preparation of the cell, counterbalance, and rotor, methods of calculation, and maintenance of optical components are essentially the same as for the model E analytical ultracentrifuge (1). After the initial alignment of the accessory no further alignment checks are necessary except when the lenses are cleaned after continued use. This process is simple and can be easily accomplished by the operator. EXPERIMENTAL

With the model E ultracentrifuge as a standard, experiments were made to determine sedimentation coefficients and molecular weights of known macromolecules. The rotor and cells used to accomplish these measurements were the same as would be used in the model E. Sample concentrations for the sedimentation velocity runs were 10 mg/ml and for equilibrium sedimentation runs 5 mg/ml. The shorter optical track and lower cylindrical lens magnification permitted the use of these concentrations for the best viewing of the schlieren image.

222

GRIFFITH

AND

GROPPER

The samples used for sedimentation velocity runs were bovine serum albumin” (BSA) and abnormal human serum.4 Both samples were made up in 0.25 M acetate buffer, pH 4.4, and a speed of 60,000 rpm was maintained throughout both experiments. The run

FIG. 3. Schlieren photographs taken from sedimentation velocity experiment with bovine serum albumin after maximum speed was attained: (1) 40 min, (2) 80 min, (3) 100 min, (4) 120 min, (5) 140 min.

temperature was 20°C. A 4” single sector cell assembly was used and the sedimentation coefficients were calculated from the peak movements at chosen time intervals. Figure 3 shows the photographs taken from the experiment with the bovine serum albumin and Figure 4 shows those taken from the experiment with abnor-

FIG. 4. Schlieren photographs taken from sedimentation velocity experiment with abnormal human serum after maximum speed was attained : (1) at maximum speed of 60,000 rpm, (2) 15 min, (3) 25 min, (4) 40 min, (5) 55 min, (6) 70 min, (7) 85 min, (8) 100 min, (9) 115 min. s Available 4 Available

from Pentex Incorporated, from Hyland Laboratories,

Kankakee, Illinois. Los Angeles, California.

SCHLIEREN

OPTICS

223

ACCESSORY

ma1 human serum. The 0.010” wire diaphragm was used in this experiment because it provided a better view of the schlieren image. The sample used for the sedimentation equilibrium run was ribonuclease A5 (5 mg/ml) , made up in 0.25 M PO, buffer, pH 7.0. The speed used for this 18 hr run was 20,000 rpm and the temperature was controlled at 20°C. Since a plot of the slope of the concentration gradient throughout the cell is dependent on the buffer concentration, the double-sector cell was used to obtain the baseline position. This cell was filled in the same manner as is done for a similar experiment in the model E. Figure 5 shows a photograph of the schlieren pattern measured for this experiment. The 0.003” wire diaphragm was used in the equilibrium experiment because its thickness provided less discrepancies when measuring the gradient profile.

FIG. 5. Schlieren photograph after 18 hr at 20,000 rpm.

of equilibrium

run with

ribonuclease

A taken

RESULTS

Calculations for sedimentation coefficients were based on the formula : s=$ where x is the distance of the boundary or peak movement in centimeters, t is the time in seconds, and o is the angular velocity in radians per second (2). This is the same formula that is generally used when the model E is employed. In the bovine serum albumin run the sedimentation coefficient was calculated to be 4.2 S and in the abnormal human serum experiments the sedimentation of the observed peaks were 4.5, 6.9, and 5 Available sey.

from Worthington

Biochemical

Corporation,

Freehold,

New Jer-

224

GRIFFITH

AND

GROPPER

9.2 S, respectively. There was a smaller fast-moving component which was only slightly visible in the first two photographs, due to the lower magnification of the cylindrical lens as compared to that of the model E. In the sedimentation equilibrium run the calculations were based on the formula:

where R is the thermodynamic conversion factor 8.3144 X lo7 erg “C-1 mole-l, T is the absolute temperature, i is the partial specific volume, and r is the distance from the center of rotation. For a single component ideal system, a graph of (l/r) dc/dr versus r2 results in a straight line whose slope, when multiplied by RT/ti’ (l+), yields the molecular weight of the system. For heterogeneous systems the slope of this graph at any point yields the local x-average molecular weight. The experiment with ribonuclease gave a molecular weight of 14,000 + 250. Both cells used in the preparative ultracentrifuge with the schlieren optics were agitated and rerun in the model E to compare the results obtained with the preparative ultracentrifuge. The calculations indicated that the sedimentation coefficients and molecular weight estimates agreed within 3% when comparing the results of the two instruments. Table 1 shows the differences in performance between the schlieren optics accessory and the precision of the model E. DISCUSSION

When the albumin sample was placed in a double-sector cell and run in the model E, a small amount of dimer, approximately 2076, was observed and measured. This could not be observed with the preparative ultracentrifuge schlieren accessory. The use of a higher sample concentration (15 mg/ml) in the cell proved futile because the light was deviated out of the optical path at this concentration. In the serum sample the fast-moving 19 S component was observed in the first four photographs taken, whereas it was observed only in the first two photographs of the schlieren accessory. The sensitivity of the schlieren optics in the model E provides a better image of the 19 S component due to the greater magnification of the cylindrical lens. The longer optical track of the model E also enhances the observation of lower concentration components. Experiments were attempted with the three-channel equilibrium

QFormula weight

13,683.

2. Sedimentation equilibrium (a) 0.5% ribonucleasee (long-column cell and 3 mm column techniques) (b) (.2%ribonuclease meniscus depletion technique) (c) 0.2% ribonuclease (meniscus depletion technique, Rayleigh interference patterns)

Yes

80"

Yes

angle

80”

bar

gradient

Yes

ObS.

Cone.

Yes (4 components)

serum

(b) 1.0% abnormal

Obs.

Yes (2 components)

of Experiment

1. Sedimentation velocity (a) 1.0% BSA

Type

Model and

talc.

(S units)

Mol.

13,700

13,200

13,580

150

eslc.

f 250

f 300

f

wt. estimate

Obs.

bar

gradient angle

70”

80”

Not available

Yes

Yes

Obs.

Cont.

Yes (4 components)

and

eale.

(S unite)

Mol.

talc.

f

500

f 250

Not available

15,900

14,000

wt. estimate

4.2-main component (5 y0 dimer contaminant measured from area under the two peaks) 4.5, 6.9, 9.2 (resolution poor on other component)

Measured

Optics Accessory Schlieren optics accessory:peak movement

Yes (1 component)

Model E and Schlieren

1

4.1-main component (20 y0 dimer contaminant measured from area under the two peaks) 4.4, 6.7, 9 .O, 1’7.8

Measured

movement

between

E: peak

Differences in Performance

TABLE

226

GRIFFITH

AND

GROPPER

cell used in the meniscus depletion technique of Yphantis (3). The schlieren patterns were measured and the results compared with a similar experiment in the model E. The molecular weight estimates from photograph measurements agreed within 20% when the results of the two instruments were compared. Similar observations were noted when the short-column four-channel cell designed by Yphantis was used (4). The long-column equilibrium cell using step concentrations for rapid sedimentation equilibrium was employed successfully (5) . Finally, the schlieren optical system depends upon the accurate rotation of the cylindrical lens. In the schlieren optics accessory the focusing of this lens is most important for accurate molecular weight estimations because of the shorter optical track. SUMMARY

The schlieren optical accessory was designed and tested for use with the Beckman models L and L2-65B preparative ultracentrifuges. It has been used to determine sedimentation coefficients and molecular weight estimates. The optics are of sufficient quality to produce good photographs. Comparable experiments using the model E indicated that the optical accessory cannot be considered equivalent in precision to the analytical ultracentrifuge. ACKNOWLEDGMENTS We express our thanks to C. R. McEwen and C. H. Chervenka, Division Beckman Instruments, for their helpful discussions.

Spinco

REFERENCES 1. Instruction Manual, Beckman Model E Analytical Ultracentrifuge E-IM-3, May 1964. 2. SCHACHMAN, H. K., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 4, p. 32. Academic Press, New York, 1957. 3. YPHANTIS, D. A., Biochemistry 3, 29’7 (1964). 4. YPHANTIS, D. A., Ann. N. Y. Acad. Sci. 88, 586 (1960). 5. GRIFFITH, 0. M., Anal. Biochem. 19, 243 (1967).