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The molecular weights of ribonuclease and bovine plasma albumin
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
63, 247-254
(1956)
The Molecular Weights of Ribonuclease and Bovine Plasma Albumin1 Stanley M. Klainer” and Gerson Kegeles From l’he Department of Chemistry, Clark Worcester, Massachusetts Received
February
University/,
24, 1956
Among those proteins whose molecular weight,s have been the subject of controversy, ribonuclease is a not’able example (1). ,4 reinvestigation was instituted by the classical methods of sedimentat’ion and diffusion,” and difficulties in sedimenting this protein free from the meniscus in available centrifugal fields mere responsible in part for initiat#ing work to form boundaries by flow techniques in the rotating ultracentrifuge wll (2) . Marc recently, applications of the suggestions by Awhibald (3) for obtaining molecular weights during the approach to sedimentation cquilibrium have been standardized with known small molecules, meeting with very encouraging success (4-G). The present’ paper represents our initial efforts to restudy in detail a series of highly purified proteins, wit)h a view to est)ablishing molecular weight data free from rertain difficulties and errors inherent in diffusion measurements on some highly purified proteins. It’ is felt that the success of these studies on rihonuclease and bovine plasma albumin indicaks the power and (saw of applicabilit)y of the Archibald ultracentrifuge met)hod to proteins.
As our method of application of Arrhibald’a suggest,ions has already been out,lined in considerable detail (G), only enough descript,ion of the 1 This work was made possible by U. S. Public Health Service Grant RG-3449. * To be presented by Stanley M. Klainer to the faculty of Clark University in partial fulfillment of the requirements for the Ph.D. degree. 3 Sorof, S., and Kegeles, G., unpublished, 1951. 247
248
STANLEY
M.
KLAINER
AND
GERSON
KEGELES
methodology will be outlined here to make the present applications understandable. Archibald (3) has indicated that from the time of commencement of rotation onward, the sedimentation equilibrium equation holds at the top and bottom of a liquid column containing an ideal solution of sedimentable solute : RT M
= (1 -
dC/dX
Vp)w2
xc
Here M is the gram molecular weight, R the gas constant, T the absolute temperature, V the partial specific volume of solute, P the density of solution, w the angular velocity, c the solute concentration, and x the distance measured from the center of rotation to the top or bottom of the liquid column, where the values of c and dc/dx must be determined. In a cylindrical lens schlieren optical system (7, 8), a quantity proportional to the concentration gradient dc/dx is recorded directly. The corresponding concentration c is obtained from the relations (6) C,, = Co - (l/z:)
sx x2(dc/dx) dx 20
(2)
for the t,op of the column (z = x0), and C,, = Co + (l/x:)
lb
x2(dc/dx) dx
(3)
for the bottom of the column (x = xb). The symbol X denotes the ‘rplateau region,” where the concentration is independent of the distance from the center of rotation at any given time. Here the solute concentration C,, is that originally placed in the cell. Its value is obtained most conveniently by integrating under the diagram produced at identical optical sensitivity by layering solvent over solution at very low speed in a cell employing a flow technique. (2, 6, 9): Co = lzb (dc/‘dx) dx 20
(4)
In practice all integrals have been calculated by tabular integration of data measured from schlieren diagrams with a two-coordinate microcomparator.4 4 David W. Mann Precision Instruments,
Lincoln, Mass.
MOLECULAR
WEIGHT
DETERMINATIONS
248
In lining up photographs in the microcomparator, it has been found advantageous to align the photographs so that reference marks obtained from two holes in the balancing cell bracketing the radii of the liquid column have identical readings in the dc/dx direction. For highest precision, plots of the two coordinates are extrapolated to the center position of the meniscus image. The extrapolated vertical (dc/ rlr) coordinate so obtained is further corrected by adjusting the average vertical coordinate to zero in the ‘Lplateau region.” In the event that the solvent contains sedimentable salts, buffer reference phot’ographs taken at the same speeds and times of centrifugation must he used for comparison. When C,, and C,, of Eqs. (2) and (3) are int’roduced into Eq. (l), it is. necessary to multiply the tabular integrals corresponding to Eqs. (2), (3), and (4) by a scale factor i/F, i being the microcomparator interval along the (magnified) x axis on the photographic plate and F the camera lens enlargement. This stems from the fact that there is no corresponding factor in dc/dx in the numerator of Eq. (1). All other factors such as the instrument opt’ical constants and the specific refraction of solute cancel out.. EXPERIMENTAL
The experiments were performed in a Spinco model E ultracentrifuge equipped with a phase-contrast schlieren diaphragm having a human hair glued t,o the phase border to define the base-line region (6, 10-12). Throughout this work, as well as the previous report (6), the diaphragm has been kept at 80” with respect to the source slit image. This relatively low sensitivity has been found to be advantageous in keeping the curve outlines sharp and in minimizing the uncertainty of the curve position at the meniscus. Crystalline ribonuclease (bovine), lot No. 381.59, was obtained from Armour and Company. Solutions of this protein whose concentrations varied from 0.812 to 0.025% were made by dissolving the ribonuclease directly in a buffer consisting of 0.1 M sodium chloride, 0.004 M potassium dihydrogen phosphate, and 0.035 /U dipotassium hydrogen phosphate at pH 7.8. These solutions were dialyzed against, three changes of buffer over a period of 24 hr. at 0°C. just before each experiment, except that solutions of concentration below 0.05% were not dialyzed. Solutions were kept cold until the ultracentrifuge cell was filled, to avoid any possibility of enzymatic degradation of the protein. The partial specific volume of ribonuclease \vas taken from the measurement.s of Rothen (1) as 0.709 ct./g. at 25°C. In this work it was found that t,he solutions containing 0.025% ribonuclease gave insufficient refractive-index gradients at the top of the column to permit measurement of molecular weight,, and molecular weights at this concentrat,ion were derived from the bottom of the column only.
250
STANLEY
R
M.
A
KLAINER
AND
GERSON
S
-_
KEGELES
C
R
_-
:
..
--
1
FIG. 1. Typical ultracentrifuge diagram with solution-air meniscus and solution-carbon tetrachloride meniscus. Key: R, reference; A, air; S, solution; C, carbon tetrachloride.
It was also found impossible to measure Co directly with the aid of the boundary-forming cell at the lowest concentrations. Therefore these solutions were prepared accurately by weight without subsequent dialysis, and a similarly prepared 1% solution was used for this measurement, one-fortieth of this Co integral being used at O.O25oJ,, for example. Bovine plasma albumin6 solutions were prepared by dissolving phosphorus pentoxide-desiccated Armour’s crystalline lot No. 370295-A in a buffer consisting of 0.15 Msodium chloride, 0.02 M sodium acetate, and 0.03 2M acetic acid at pH 4.40. This protein was previously (13) found to be electrophoretically isoelectric in this buffer. These solutions were also dialyzed against several changes of buffer for 24 hr. before use. The partial specific volume for bovine plasma albumin was taken from the work of Dayhoff, Perlmann, and MacInnes (14) as 0.7343 at 25°C. Considerable information concerning homogeneity might be obtained by studying the molecular weight at both the top and bottom of the cell (3). As the cell bottom has been difficult to locate (5,6), a valuable suggestion has been made to form a second visible meniscus between aqueous solution and carbon tetrachloride near the cell bottom.6 In the standard 4” sector cell, 0.1 ml. of carbon tetrachloride is a convenient quantity to make the bottom of the aqueous column visible (Fig. 1). RESULTS
In Table I are summarized the molecular weight results obtained from four separate experiments at different concentrations of ribonuclease. It is noted that by increasing the speed of centrifugation, it becomes pos5 We are indebted to Armour Research Laboratories for a gift of this sample in experimental quantities. 8 We are indebted to Dr. Howard K. Schachman for this suggestion, and for also pointing out that in the standard Spinco ultracentrifuge cells, the bottom does not follow the arc generated at the center of rotation.
MOLECULAR
WEIGHT
TABLE Molecular nt.
per cent
solute.
5
251
DETERMINATIOKS
I
Weight of Ribonuclease
0.811
0.532
0.2xfl
0.025
210
337
SYi
524
-. Speed,
rezl./sec. Time,
min.
I
1
Top portion I
of cell
16
I
14,000
32 48
I1
13,900
~
13,700 14,300
Ii
13,800
i
13,800
~
64
Bottom
14,100
portion
14,300
1
-
13,800 14,100
~
-
,
of cell
16
-
14,000
14,200
/
-
32 48 64
-
14,000 14) 300 14) 300
13,700 13,800 13,900
! I j
13,700 14,000
sible to make measurements at successively lower concentrations, until, for essentially uncharged globular protein molecules, there is no further question of contributions from nonideality of solutions. The small effect of nonideality is indicat,ed by the close agreement’ between values at 0.812 and 0.025 %. It is noted that no significant difference appears between results obtained at the top and bottom of the aqueous liquid column. Moreover, no significant drift occurs with time. Within the 64 min. of observation, it is concluded that we find no evidence of inhomogeneit’y in molecular weight. Ot’her evidence of molecular inhomogeneity of highly purified ribonuclease preparations (15, 16) is an indicat’ion of subtle differences in surface arrangement of charged groups. From our measurements, we estimate the molecular weight of ribonuclease, referred to the anhydrous state (17) for which the partial specific volume was measured (1) to be 14,000 f 200. This may be compared with t’he sedimentation-diffusion molecular weight of approximately 13,000 given by Rothen (l), and with the x-ray molecular weight of 13,400 reported by Carlisle and Scouloudi (18), and with the chemically determined value of 13,895 reported by Hirs, Moore,
252
STANLEY
M.
Molecular wt.
per cent solute,
Speed,
%
I
KLAINER
AND
GERSON
KEGELES
TABLE II Weight of Bovine Plasma Albumin 1.017
0.505
I
I
0.108
rev./set. 133.6 Time,
131.5
203.2
71,000 70,ooo
71,000 71,000
min.
Top portion of cell 32 64
70,ooo 70,000
Bottom portion of cell 32 64
70,000 71,000
70,000 70,000
69,000 71,ooo
and Stein (16). By sedimentation measurements with a Spinco syntheticboundary cell (9) and diffusion measurements using the Rayleigh method (19), Ginsburg and Schachman’ find a best value of 14,000 for the molecular weight. In Table II are shown values for the molecular weight of bovine plasma albumin determined from the top and bottom of the cell at three concentrations. The average of 70,300 f 600 from these measurements compares with our previous average (6) of 71,300 from two sets of measurements at the top of the cell only. Additional comparison may be made with the value of 67,300 computed with the diffusion data of Akeley and Gosting (20) and the temperature-corrected (21, 22) sedimentation data of Kegeles and Gutter (13). Calculation with the diffusion data of Hoch (23), however, leads to a molecular weight over 71,000. DISCUSSION
It is interesting to point out the contrast between the consistency of sedimentation velocity measurements of various investigators for different preparations of bovine plasma albumin (24) and the inconsistency of diffusion measurements of at least equally high internal precision under similar circumstances (20, 23). This must be attributed to small, but varying amounts of large aggregates, which are by the nature of the ex7 Ginsburg, A., and Schachman, H. K., private communication.
MOLECULAR
WEIGHT
DETERMINATIONS
253
periment dropped out of consideration in sedimentation experiments, although they weigh very heavily in diffusion experiments. Such considerations serve further to indicate the potential value of Archibald’s direct approach to the molecular weight. SUMMARY
With the methodology outlined, it now appears possible, by use of the Archibald procedure, to obtain a reliable value for the molecular weight of a protein within an hour of sedimentation in the ultracentrifuge. The ult,racentrifugal data so obtained need be supplemented only by a measurement of partial specific volume. As previously demonstrated, the same method also holds through the whole molecular weight rangedown to 504, and recent data of Wade (25) obtained in this laboratory for glycyl-Lleucine indicate equal precision for a molecular weight of only 188. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
17. 18. 19.
ROTHEN, A., J. Gen. Physiol. 24, 203 (1940). KEGELES, G., J. Am. Chem. Sot. 74, 5532 (1952). ARCHIBALD, W. J., J. Phys. & Colloid Chem. 61, 1204 (1947). PORATH, J., Acta Chem. Stand. 6, 1237 (1952). BROWN, R. A., KRITCHEVSKY, D., AND DAVIES, M., J. Am. Chem. Sot. 76,3342 (1954). KLAINER, S. M., AND KEGELES, G., J. Phys. Chem. 69, 952 (1955). PHIZPOT, J. ST. L., Nature 141, 283 (1938). SVENSSON, H., Kolloid-2. 87, 181 (1939). PICKELS, E. G., HARRINGTON, W. F., AND SCHACH&IAN, H. K., Proc. Natl. Acad. Sci. U. S. 38, 943 (1952). WOLTER, H., Ann. Physik 7, 182 (1950). ARMBRUSTER, O., KOSSEL, W., AND STROHMEIER, K., 2. Naturjorsch. 6a, 509 (1951). TRATITMAN, R., AND BURNS, V. W., Biochim. et Biophys. Acta 14, 26 (1954). KEGELES, G., AND GUTTER, F. J., J. Am. Chem. Sot. 73, 3770 (1951). DAYHOFF, M. O., PERLMANN, G. E., AND MACIXNES, D. A., J. Am. Chem. Sot. 74, 2515 (1952). MARTIN, A. J. I’., AND PORTER, R. R., Biochem. J. 49, 215 (1951). HIRS, C. H. W., STEIN, W. H., AND MOORE, S., J. Am. Chem. Sot. 73,1893 (1951); HIRS, C. H. W., MOORE, S., AND STEIN, W. H., J. Biol. Chem. 200, 493 (1953); 211, 941 (1954). GOLDBERG, R. J., J. Phys. & Colloid Chem. 67, 194 (1953). CARLISLE, C. H., AND SCOULOUDI, H., Proc. ZZoy. Sot. (London) A207, 496 (1951). SVENSSOX, H., Acta Chem. Stand. 6, 72 (1951); LONGSWORTH, L. G., J. Am. Chem. Sot. 74, 4155 (1952).
254
STANLEY
M. KLAINER
AND
GERSON
KEGELES
20. AHELEY, D. F., AND GOSTING, L. J., J. Am. Chem. Sot. 76, 5685 (1953). 21. WAUOH, D. F., AND YPHANTIS, D. A., Rev. Sci. In&. 23, 609 (1952). 22. BIANCHERIA, A., AND KEGELES, G., J. Am. Chem. Sot. 76, 3737 (1954). 23. HOCH, H., Arch. Biochem. and Biophyls. 63, 387 (1954). 24. SHULMAN, S., Arch. Biochem. and Biophys. 44, 230 (1953). 25. WADE, R., WINITZ, M., AND GREENSTEIN, J. P., J. Am. Chem. Sot.
78, 373 (1956).
OF BIOCHEMISTRY
AND
BIOPHYSICS
63, 247-254
(1956)
The Molecular Weights of Ribonuclease and Bovine Plasma Albumin1 Stanley M. Klainer” and Gerson Kegeles From l’he Department of Chemistry, Clark Worcester, Massachusetts Received
February
University/,
24, 1956
Among those proteins whose molecular weight,s have been the subject of controversy, ribonuclease is a not’able example (1). ,4 reinvestigation was instituted by the classical methods of sedimentat’ion and diffusion,” and difficulties in sedimenting this protein free from the meniscus in available centrifugal fields mere responsible in part for initiat#ing work to form boundaries by flow techniques in the rotating ultracentrifuge wll (2) . Marc recently, applications of the suggestions by Awhibald (3) for obtaining molecular weights during the approach to sedimentation cquilibrium have been standardized with known small molecules, meeting with very encouraging success (4-G). The present’ paper represents our initial efforts to restudy in detail a series of highly purified proteins, wit)h a view to est)ablishing molecular weight data free from rertain difficulties and errors inherent in diffusion measurements on some highly purified proteins. It’ is felt that the success of these studies on rihonuclease and bovine plasma albumin indicaks the power and (saw of applicabilit)y of the Archibald ultracentrifuge met)hod to proteins.
As our method of application of Arrhibald’a suggest,ions has already been out,lined in considerable detail (G), only enough descript,ion of the 1 This work was made possible by U. S. Public Health Service Grant RG-3449. * To be presented by Stanley M. Klainer to the faculty of Clark University in partial fulfillment of the requirements for the Ph.D. degree. 3 Sorof, S., and Kegeles, G., unpublished, 1951. 247
248
STANLEY
M.
KLAINER
AND
GERSON
KEGELES
methodology will be outlined here to make the present applications understandable. Archibald (3) has indicated that from the time of commencement of rotation onward, the sedimentation equilibrium equation holds at the top and bottom of a liquid column containing an ideal solution of sedimentable solute : RT M
= (1 -
dC/dX
Vp)w2
xc
Here M is the gram molecular weight, R the gas constant, T the absolute temperature, V the partial specific volume of solute, P the density of solution, w the angular velocity, c the solute concentration, and x the distance measured from the center of rotation to the top or bottom of the liquid column, where the values of c and dc/dx must be determined. In a cylindrical lens schlieren optical system (7, 8), a quantity proportional to the concentration gradient dc/dx is recorded directly. The corresponding concentration c is obtained from the relations (6) C,, = Co - (l/z:)
sx x2(dc/dx) dx 20
(2)
for the t,op of the column (z = x0), and C,, = Co + (l/x:)
lb
x2(dc/dx) dx
(3)
for the bottom of the column (x = xb). The symbol X denotes the ‘rplateau region,” where the concentration is independent of the distance from the center of rotation at any given time. Here the solute concentration C,, is that originally placed in the cell. Its value is obtained most conveniently by integrating under the diagram produced at identical optical sensitivity by layering solvent over solution at very low speed in a cell employing a flow technique. (2, 6, 9): Co = lzb (dc/‘dx) dx 20
(4)
In practice all integrals have been calculated by tabular integration of data measured from schlieren diagrams with a two-coordinate microcomparator.4 4 David W. Mann Precision Instruments,
Lincoln, Mass.
MOLECULAR
WEIGHT
DETERMINATIONS
248
In lining up photographs in the microcomparator, it has been found advantageous to align the photographs so that reference marks obtained from two holes in the balancing cell bracketing the radii of the liquid column have identical readings in the dc/dx direction. For highest precision, plots of the two coordinates are extrapolated to the center position of the meniscus image. The extrapolated vertical (dc/ rlr) coordinate so obtained is further corrected by adjusting the average vertical coordinate to zero in the ‘Lplateau region.” In the event that the solvent contains sedimentable salts, buffer reference phot’ographs taken at the same speeds and times of centrifugation must he used for comparison. When C,, and C,, of Eqs. (2) and (3) are int’roduced into Eq. (l), it is. necessary to multiply the tabular integrals corresponding to Eqs. (2), (3), and (4) by a scale factor i/F, i being the microcomparator interval along the (magnified) x axis on the photographic plate and F the camera lens enlargement. This stems from the fact that there is no corresponding factor in dc/dx in the numerator of Eq. (1). All other factors such as the instrument opt’ical constants and the specific refraction of solute cancel out.. EXPERIMENTAL
The experiments were performed in a Spinco model E ultracentrifuge equipped with a phase-contrast schlieren diaphragm having a human hair glued t,o the phase border to define the base-line region (6, 10-12). Throughout this work, as well as the previous report (6), the diaphragm has been kept at 80” with respect to the source slit image. This relatively low sensitivity has been found to be advantageous in keeping the curve outlines sharp and in minimizing the uncertainty of the curve position at the meniscus. Crystalline ribonuclease (bovine), lot No. 381.59, was obtained from Armour and Company. Solutions of this protein whose concentrations varied from 0.812 to 0.025% were made by dissolving the ribonuclease directly in a buffer consisting of 0.1 M sodium chloride, 0.004 M potassium dihydrogen phosphate, and 0.035 /U dipotassium hydrogen phosphate at pH 7.8. These solutions were dialyzed against, three changes of buffer over a period of 24 hr. at 0°C. just before each experiment, except that solutions of concentration below 0.05% were not dialyzed. Solutions were kept cold until the ultracentrifuge cell was filled, to avoid any possibility of enzymatic degradation of the protein. The partial specific volume of ribonuclease \vas taken from the measurement.s of Rothen (1) as 0.709 ct./g. at 25°C. In this work it was found that t,he solutions containing 0.025% ribonuclease gave insufficient refractive-index gradients at the top of the column to permit measurement of molecular weight,, and molecular weights at this concentrat,ion were derived from the bottom of the column only.
250
STANLEY
R
M.
A
KLAINER
AND
GERSON
S
-_
KEGELES
C
R
_-
:
..
--
1
FIG. 1. Typical ultracentrifuge diagram with solution-air meniscus and solution-carbon tetrachloride meniscus. Key: R, reference; A, air; S, solution; C, carbon tetrachloride.
It was also found impossible to measure Co directly with the aid of the boundary-forming cell at the lowest concentrations. Therefore these solutions were prepared accurately by weight without subsequent dialysis, and a similarly prepared 1% solution was used for this measurement, one-fortieth of this Co integral being used at O.O25oJ,, for example. Bovine plasma albumin6 solutions were prepared by dissolving phosphorus pentoxide-desiccated Armour’s crystalline lot No. 370295-A in a buffer consisting of 0.15 Msodium chloride, 0.02 M sodium acetate, and 0.03 2M acetic acid at pH 4.40. This protein was previously (13) found to be electrophoretically isoelectric in this buffer. These solutions were also dialyzed against several changes of buffer for 24 hr. before use. The partial specific volume for bovine plasma albumin was taken from the work of Dayhoff, Perlmann, and MacInnes (14) as 0.7343 at 25°C. Considerable information concerning homogeneity might be obtained by studying the molecular weight at both the top and bottom of the cell (3). As the cell bottom has been difficult to locate (5,6), a valuable suggestion has been made to form a second visible meniscus between aqueous solution and carbon tetrachloride near the cell bottom.6 In the standard 4” sector cell, 0.1 ml. of carbon tetrachloride is a convenient quantity to make the bottom of the aqueous column visible (Fig. 1). RESULTS
In Table I are summarized the molecular weight results obtained from four separate experiments at different concentrations of ribonuclease. It is noted that by increasing the speed of centrifugation, it becomes pos5 We are indebted to Armour Research Laboratories for a gift of this sample in experimental quantities. 8 We are indebted to Dr. Howard K. Schachman for this suggestion, and for also pointing out that in the standard Spinco ultracentrifuge cells, the bottom does not follow the arc generated at the center of rotation.
MOLECULAR
WEIGHT
TABLE Molecular nt.
per cent
solute.
5
251
DETERMINATIOKS
I
Weight of Ribonuclease
0.811
0.532
0.2xfl
0.025
210
337
SYi
524
-. Speed,
rezl./sec. Time,
min.
I
1
Top portion I
of cell
16
I
14,000
32 48
I1
13,900
~
13,700 14,300
Ii
13,800
i
13,800
~
64
Bottom
14,100
portion
14,300
1
-
13,800 14,100
~
-
,
of cell
16
-
14,000
14,200
/
-
32 48 64
-
14,000 14) 300 14) 300
13,700 13,800 13,900
! I j
13,700 14,000
sible to make measurements at successively lower concentrations, until, for essentially uncharged globular protein molecules, there is no further question of contributions from nonideality of solutions. The small effect of nonideality is indicat,ed by the close agreement’ between values at 0.812 and 0.025 %. It is noted that no significant difference appears between results obtained at the top and bottom of the aqueous liquid column. Moreover, no significant drift occurs with time. Within the 64 min. of observation, it is concluded that we find no evidence of inhomogeneit’y in molecular weight. Ot’her evidence of molecular inhomogeneity of highly purified ribonuclease preparations (15, 16) is an indicat’ion of subtle differences in surface arrangement of charged groups. From our measurements, we estimate the molecular weight of ribonuclease, referred to the anhydrous state (17) for which the partial specific volume was measured (1) to be 14,000 f 200. This may be compared with t’he sedimentation-diffusion molecular weight of approximately 13,000 given by Rothen (l), and with the x-ray molecular weight of 13,400 reported by Carlisle and Scouloudi (18), and with the chemically determined value of 13,895 reported by Hirs, Moore,
252
STANLEY
M.
Molecular wt.
per cent solute,
Speed,
%
I
KLAINER
AND
GERSON
KEGELES
TABLE II Weight of Bovine Plasma Albumin 1.017
0.505
I
I
0.108
rev./set. 133.6 Time,
131.5
203.2
71,000 70,ooo
71,000 71,000
min.
Top portion of cell 32 64
70,ooo 70,000
Bottom portion of cell 32 64
70,000 71,000
70,000 70,000
69,000 71,ooo
and Stein (16). By sedimentation measurements with a Spinco syntheticboundary cell (9) and diffusion measurements using the Rayleigh method (19), Ginsburg and Schachman’ find a best value of 14,000 for the molecular weight. In Table II are shown values for the molecular weight of bovine plasma albumin determined from the top and bottom of the cell at three concentrations. The average of 70,300 f 600 from these measurements compares with our previous average (6) of 71,300 from two sets of measurements at the top of the cell only. Additional comparison may be made with the value of 67,300 computed with the diffusion data of Akeley and Gosting (20) and the temperature-corrected (21, 22) sedimentation data of Kegeles and Gutter (13). Calculation with the diffusion data of Hoch (23), however, leads to a molecular weight over 71,000. DISCUSSION
It is interesting to point out the contrast between the consistency of sedimentation velocity measurements of various investigators for different preparations of bovine plasma albumin (24) and the inconsistency of diffusion measurements of at least equally high internal precision under similar circumstances (20, 23). This must be attributed to small, but varying amounts of large aggregates, which are by the nature of the ex7 Ginsburg, A., and Schachman, H. K., private communication.
MOLECULAR
WEIGHT
DETERMINATIONS
253
periment dropped out of consideration in sedimentation experiments, although they weigh very heavily in diffusion experiments. Such considerations serve further to indicate the potential value of Archibald’s direct approach to the molecular weight. SUMMARY
With the methodology outlined, it now appears possible, by use of the Archibald procedure, to obtain a reliable value for the molecular weight of a protein within an hour of sedimentation in the ultracentrifuge. The ult,racentrifugal data so obtained need be supplemented only by a measurement of partial specific volume. As previously demonstrated, the same method also holds through the whole molecular weight rangedown to 504, and recent data of Wade (25) obtained in this laboratory for glycyl-Lleucine indicate equal precision for a molecular weight of only 188. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
17. 18. 19.
ROTHEN, A., J. Gen. Physiol. 24, 203 (1940). KEGELES, G., J. Am. Chem. Sot. 74, 5532 (1952). ARCHIBALD, W. J., J. Phys. & Colloid Chem. 61, 1204 (1947). PORATH, J., Acta Chem. Stand. 6, 1237 (1952). BROWN, R. A., KRITCHEVSKY, D., AND DAVIES, M., J. Am. Chem. Sot. 76,3342 (1954). KLAINER, S. M., AND KEGELES, G., J. Phys. Chem. 69, 952 (1955). PHIZPOT, J. ST. L., Nature 141, 283 (1938). SVENSSON, H., Kolloid-2. 87, 181 (1939). PICKELS, E. G., HARRINGTON, W. F., AND SCHACH&IAN, H. K., Proc. Natl. Acad. Sci. U. S. 38, 943 (1952). WOLTER, H., Ann. Physik 7, 182 (1950). ARMBRUSTER, O., KOSSEL, W., AND STROHMEIER, K., 2. Naturjorsch. 6a, 509 (1951). TRATITMAN, R., AND BURNS, V. W., Biochim. et Biophys. Acta 14, 26 (1954). KEGELES, G., AND GUTTER, F. J., J. Am. Chem. Sot. 73, 3770 (1951). DAYHOFF, M. O., PERLMANN, G. E., AND MACIXNES, D. A., J. Am. Chem. Sot. 74, 2515 (1952). MARTIN, A. J. I’., AND PORTER, R. R., Biochem. J. 49, 215 (1951). HIRS, C. H. W., STEIN, W. H., AND MOORE, S., J. Am. Chem. Sot. 73,1893 (1951); HIRS, C. H. W., MOORE, S., AND STEIN, W. H., J. Biol. Chem. 200, 493 (1953); 211, 941 (1954). GOLDBERG, R. J., J. Phys. & Colloid Chem. 67, 194 (1953). CARLISLE, C. H., AND SCOULOUDI, H., Proc. ZZoy. Sot. (London) A207, 496 (1951). SVENSSOX, H., Acta Chem. Stand. 6, 72 (1951); LONGSWORTH, L. G., J. Am. Chem. Sot. 74, 4155 (1952).
254
STANLEY
M. KLAINER
AND
GERSON
KEGELES
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