THE JOURNAL

OFBro~oorcar. CHEMISTRY
Vol. 236, No. 5, May 1961
P&&d in U.S.A.

A Method for Determining the Sedimentation Behavior

of Enzymes: Application to Protein Mixtures
ROBERT G. MARTIN* AND BRUCE N. AMES

From the National Institute of Arthritis and Metabolic Diseases,National Institutes of Health,
United States Public Health Service, Bethesda, Maryland

(Received for publication, December 5, 1960)

During an investigation of the gene-enzyme relationships in EXPERIMENTAL PROCEDURE

histidine biosynthesis in Xalmonella typhimurium, it became Materials and AssaysLyophilized yeast alcohol dehydro-
desirable to determine, in crude extracts, the approximate mo- genase obtained from Worthington Biochemical Corporation
lecular weights of several enzymes. We have found sucrose was dissolved in 0.05 M Tris-HCl buffer, pH 7.5, to a concentra-
gradient centrifugation to be a suitable method for determining tion of 10 mg per ml and stored at 3”. Before use on the sucrose
sedimentation coefficients of enzymes in protein mixtures. A gradient it was diluted to 0.20 mg per ml with the Tris buffer.

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variety of enzymes of known properties have been studied in the The dehydrogenase was assayed in a Cary spectrophotometer
development of the method. by following the increase in absorption at 340 rnp for 20 seconds
Although the separation cell of Yphantis and Waugh (1) has of a l-ml reaction mixture containing 170 pmoles of ethanol, 50
been demonstrated to be applicable to the determination of pmoles of Tris, pH 8.5, 15 pmoles of DPN, and 5 to 20 ~1 of
molecular weights when multiprotein solutions are used, the enzyme fraction. Units of activity were expressed in terms of
present method has several advantages over that system and change in absorbancy per 20 seconds per 10 ~1 of enzyme frac-
these advantages will be discussed. tion.
Sucrose gradient centrifugation, using the swinging bucket Lyophilized egg white lysozyme (Worthington, twice crystal-
head of the preparative ultracentrifuge, has been used exten- lized) was dissolved in 0.05 M Tris buffer, pH 7.5, to a concen-
sively in the determination of sedimentation constants of vi- tration of 100 mg per ml and stored at 3”. Before use it was
ruses, mitochondria, microsomes, and ribosomes (2, 3). The diluted to 5 mg per ml in this buffer. Lysozyme was assayed
adaptation of this method to relatively low molecular weight by following the decrease in turbidity at 650 rnp of a l-ml reac-
substances is reported here. tion mixture containing 10 pmoles of Tris, pH 8.0, and enough
In the sucrose gradient technique the sample to be studied is Micrococcus Zysodeikticus cell walls (Difco Laboratories, Bacto-
layered on a gradient and materials of different sedimentation lysozyme substrate) to give an absorbancy of approximately 2.0
properties separate from each other during the centrifugation. absorbancy units in the Cary spectrophotometer. The reaction
A hole is then punched in the bottom of the centrifuge tube and was started with 5 to 20 ~1 of enzyme fraction and activity was
fractions are collected and analyzed. expressed in terms of the change in absorbancy per 20 seconds
With slight modifications in the design of the apparatus for per 10 ~1 of enzyme fraction.
gradient production and fractionation, we have found the Beef liver catalase was obtained as an aqueous ammonium
method to be simple and accurate. Sedimentation coefficients sulfate suspension of approximately 40 mg per ml of protein
have been determined for a number of well characterized en- (Worthington). Before use, it was diluted to 0.40 mg per ml
zymes as well as a sample of ribonucleic acid, and the results in 0.05 M Tris buffer, pH 7.5. Catalase was assayed by follow-
are in good agreement with the values reported by others. The ing the decrease in absorbancy at 240 rnp of a 3-ml reaction
same values have been obtained whether the enzymes were mixture containing 30 amoles of potassium phosphate buffer
analyzed as pure proteins or mixed with crude extracts. at pH 7.5, 18 pmoles of HzOl, and 5 to 20 ~1 of enzyme fraction.
Although sedimentation coefficients were directly calculated Activities were calculated in terms of change in absorbancy per
for a variety of substances in order to determine the accuracy of 20 seconds per 5 ~1 of enzyme fraction.
the method, in general use the procedure may be simplified. All enzyme assays were approximately linear in the concen-
The sedimentation coefficient (or approximate molecular weight) tration ranges used.
of an unknown enzyme may be determined by a simple ratio of Soluble RNA from rabbit liver in a concentration of approxi-
mobilities when a standard well characterized enzyme has been mately 6.5 mg per ml (130 absorbancy units per ml at 260 rnp)
added to the protein mixture. was kindly supplied by Dr. Samuel Luborsky. The RNA was
With the use of this technique the sedimentation behavior of stored at -15” and diluted with 2 volumes of water before use.
several of the enzymes in the pathway of histidine biosynthesis It was assayed by its absorption at 260 rnp.
in S. typhimurium has been determined. Extracts and Assays of His&line BiosyntheticEnzymes-The
histidine mutants were obtained from Dr. P. E. Hartman. The
* This work was begun during service as an officer in the United
States Public Health Service under the Commissioned Officers: medium, growth of strains, preparation of Nossal extracts, and
Student Training and Extern Program (CO-STEP). assays have been previously described (4) with the exception

1372
May 1961 R. G. Martin and B. N. Ames 1373

that histidinol was substituted for histidine in the growth media one chamber and the block rocked back and forth to free air
of the histidine-requiring mutants.1 bubbles that might have been caught in the passage between
Crude, undialyzed extracts
were usually placed directly on the sucrose gradient. the two chambers.
In some The stopcock was then turned to the closed
cases the extract was subjected to Sephadex gel filtration position and the two chambers were emptied and wiped dry.
(5)
by passing 1 ml of extract through a l- X 5-cm column contain- The block was placed in a clamp, the stirrer adjusted, the out-
ing 1 g of Sephadex G-25 (Pharmacia Company, flow tubing bent upward so that its tip was above the top of
Uppsala,
Sweden), equilibrated with 0.01 M Tris, pH 7.5. The protein the chambers, and each of the two chambers was filled with 2.3
solution was eluted with the Tris buffer, and the 1.2 ml which ml of sucrose-buffer solution of the desired concentration. In
contained the protein free from salt were collected. all the experiments reported here, 20% (weight per volume) of
In order to obtain a partial purification cold sucrose (0.584 M) in 0.05 M Tris-HCl buffer at pH 7.5 was
of the histidine en-
zymes, a DEAE-cellulose (6) column similar to that previously placed in the mixing chamber and 5% (weight per volume) of
described was used (7). SaZmonelZa hisG-46 mutant cells werecold sucrose (0.146 M) in 0.05 M Tris-HCl buffer at pH 7.5 was
grown and harvested. The cells were suspended in 10 ml of placed in the adjacent chamber. The rotor was started, the
TSM buffer2 and subjected to sonic oscillation for 10 minutes stopcock opened, and the tip of the outflow tube placed at the
at 0” in a lo-kc Raytheon sonic oscillator. top of a Lusteroid centrifuge tube. Occasionally gentle suction
The extract was
spun at 25,000 x 9 for 45 minutes and the supernatant was required to start the flow. To assure linearity of the gra-
was
passed through a 3- x 21-cm, 20-g Sephadex G-25 column which dient, care was taken to observe that the fluid levels in the two
had been equilibrated with TSM buffer. The fractions showing chambers were equal during emptying.
appreciable activity were combined. (The specific activity of In initial experiments the production of gradients was tested
this sonicated extract was approximately by mixing dichlorophenolindophenol
the same as the spe- with the 20 y0 sucrose

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cific activities obtained on the Nossal extracts,) solution.
The enzyme Perfectly linear plots of absorbancy at 600 mp against
was then purified on a 2- x 25-cm, 8.3-g gravity-packed fraction number were obtained when 44 fractions were collected.
DEAE-
cellulose column, washed according to Peterson and Sober (6), Gradients were stable for at least 48 hours. A 0.05-ml hold-up
and equilibrated with TSM buffer. The enzyme extract (20 volume in the apparatus resulted in the delivery of only 4.55
ml) was washed into the column with 5 ml of TSM buffer and ml to the centrifuge tube.
then eluted with a linear 0.00 to 0.80 M NaCl gradient in TSM Layering of Sample-The gradients were stored in a 3” cold
buffer. Fractions of approximately 2.3 ml were collected everyroom for 4 to 18 hours before use. TO start a run, the substance
90 seconds. Protein concentrations were determined according to be studied was diluted to the desired concentration and 0.10
to the method of Lowry et al. (8). As previously reported (7), ml was layered on the gradient; care was taken to avoid bubble
complete separation of histidinol dehydrogenase and imidazole formation. It is essential that the material to be layered on
acetol phosphate transaminase from each other and from the the gradient float on 5% sucrose. To insure convection-free
histidinol phosphate phosphatase-imidazole sedimentation,
glycerol phosphate protein solutions of considerably less than 5%
dehydrase peak was obtained. The ratio of phosphatase activ- (approximately 2% or less) must be used (3). A sharp inter-
ity to dehydrase activity was constant throughout face between the sucrose and protein solution was always ob-
the eluted
fractions (7). A IO-fold increase in specific activity was ob- served and this interface remained distinct for the several min-
tained for each of the enzymes. The fractions with maximal utes required to load the centrifuge tubes into the rotor buckets.
activity had a 450-fold higher specific activity than wild-type Centrifugation-The characteristics and dimensions of the
and contained less than 2.5% nucleic acid based on their 280 to swinging bucket rotor SW-39 designed to fit the model L Spinco
260 rnh ratio of 1.1 (9). centrifuge (Beckman Instruments, Inc., Spinco Division, Palo
Apparatus for Making Sucrose Gradients-A Alto, California) have been described (lo), as have the errors
modification of
the simple design of Britten and Roberts (3) was used to pro- resulting from the use of nonsector-shaped centrifuge tubes (2).
duce linear sucrose gradients. Their design consists of a block The total volume used in these experiments was 4.65 ml (4.55-ml
of Lucite containing two chambers that are connected at the gradient plus O.l-ml sample) and the distance from rotor center
bottom by a removable screw pin. An outflow tube extends to meniscus, allowing 0.01 cm for rotor stretch and 0.03 cm for
from one chamber. This basic design was altered only in that radial shift of the meniscus, was calculated to be 6.02 cm (10).
a stopcock was introduced between the two chambers to replace Further correction is required because the protein layer is not
the screw pin. We employed a polyethylene outflow tube which infinitesimally thin. It was assumed that the protein moves
was drawn out in a flame so that emptying time with 2.3 ml of from the middle of the layer. The calculated distance from the
sucrose in each chamber was approximately 10 minutes. rotor center to the middle of the protein layer was 6.06 cm.
The
chamber next to the outflow tube was stirred with a platinum Because of the stability rendered to the solution by the sucrose
bacteriological inoculating loop which was mounted on a motor. gradient (2, 3) very much less care was needed in the operation
The stirring speed was adjusted to give good mixing with mini- of the centrifuge than described by Hogeboom and Kuff (10).
mal disturbance of the meniscus. The rotor was accelerated very slowly for approximately 10
The apparatus was filled by turning the stopcock to the seconds to eliminate the initial lash given to the rotor by the
open position so that free flow existed between the two drive shaft when the two were not fully engaged. After this,
chambers. The less dense sucrose solution was then added to the r.p.m. control knob was immediately turned to full speed, a
setting of 39,000 r.p.m. The rotor was decelerated by turning
1 Specific activities for the histidine enzymes up to 40 times wild the time knob to zero and allowing the rotor to coast to a halt
type have been observed when Salmonella his mutants are grown
with the brake off.
on 0.05 rnM histidinol .
* TSM buffer: 0.01 M Tris (free base), 0.005 M magnesium ace- It is worthy of note that accurate calculations of rotor speed
ta,te, and 0.004 M succinic acid, the pH adjusted with NaOH to 7.6, must be based on the odometer readings; the speed setting and
1374 Sedimentation Behavior of Enzymes Vol. 236, No. 5

tachometer are unreliable.3 During relatively long runs (over FRACTIONATOR

4 hours), the rotor speed tends to vary somewhat. Conse-
quently, for each centrifugation the odometer was read before
starting and at the time when deceleration was begun so that an
average speed was obtained. The time was then corrected to
the equivalent time of centrifugation at 38,000 r.p.m. by the
equation, (r.p.m.)tti = (r.p.m.)$z. The time of centrifugation
was taken as the period from the start of acceleration of the THIS END OF END
rotor to the start of deceleration. Deceleration took 13 minutes
from full speed, and integration of the plot of time against speed LUSTEROIDTUBE BEING
during deceleration gave an estimated 41 minutes of centrifuga- LOWEREDINTO POSITION
tion at 38,000 r.p.m. Acceleration took 4 minutes, which was
equivalent to 24 minutes of centrifugation at 38,000 r.p.m.
Therefore, 3 minutes were added to the time of centrifugation. CUT OFF END OF
Another troublesome aspect of the centrifuge was the main- CENTRIFUGETUBE
tenance of a constant temperature during the centrifugation, BOTTOM END PIECE
this perhaps because our instrument was not equipped with an
auxiliary ultrahigh vacuum system. Successful runs at 3” in the
swinging bucket rotor were accomplished only by precooling the
rotor chamber (with the vacuum on) to its lowest setting (-18” FIG. 1. A Nalgene drying tube with two end pieces (No. 1251 B,

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on our instrument). The precooled head (3”) was then rapidly Phipps & Bird, Inc., Richmond, Virginia) was cut to 2.5 inches.
loaded into the centrifuge. With the above precautions, the A Nalgene centrifuge tube, 100 X 16 mm (No. 1210-1, Phipps &
Bird, Inc.) with an internal diameter which just allowed easy
temperature increase of the samples during 17 hours of centri- passage of the Lusteroid centrifuge tube was cut to a 1.5.inch
fugation could be kept to less than O.8o.4 hollow cylinder. This cylinder was forced into the drying tube
Sampling-In their work with CsCl gradient centrifugation, far enough to allow the bottom end piece of the drying tube to
Weigle et al. (11) emptied centrifuge tubes progressively and rest against the cylinder when the end niece was inserted in the
drying tube. The bottom end piece was-then fitted with a No. 00
uniformly by punching a hole in the bottom of each tube with a rubber stopper carefully whittled to fit snugly. On this was
needle and collecting the drops. We have devised a simple placed a circular piece of soft rubber with a diameter equal to the
fractronator, based on this principle, which yields a constant internal diameter of the end piece. A 21.gauge syringe needle
number of drops from the 4.65 ml in each tube5 (Fig. 1). In with the adapter end broken off and the sharp end filed down to a
double bevel was forced up through the rubber stopper and soft
operation, the bottom end piece with its needle was carefully
rubber gasket so that it protruded 1 mm above the latter. A
cleaned and forced into place. After the fractionator had been cork with a &mm diameter bore through it was placed in the upper
placed in a clamp, the Lusteroid centrifuge tube was carefully end piece and cut off so that it was even with the plastic ridge of
lowered into the apparatus with a pair of forceps. The upper the end piece. The upper end piece was then fitted with a rubber
end piece was positioned and pressed down, forcing the Lusteroid hose to a 50-ml syringe.
tube through the needle and starting the flow of drops. The
rate of flow could be controlled by the syringe and was kept at 0.4 ~1. As an added precaution to maintain drop size, the needle
approximately 1 drop per second. A soft rubber gasket pre- was frequently cleaned with a stylette.
vented leakage around the needle. With the system described
above, it was possible to collect 308 =t 5 drops which were usually THEORETICAL
divided into 44 fractions (7 drops in each). As the needle rested The definition of the sedimentation constant ST,,,, in a medium,
approximately 0.7 mm above the bottom of the tube, about 3 m, at temperature, T, is given by the equation (2, 13):
drops remained in the tube. To check the efficiency of the
fractionation system, a gradient was made similar to the one de-
scribed for testing the gradient-making apparatus, with dye in
the 20% sucrose. The samples were collected from the frac-
tionator and the absorbancy at 600 rnp was determined. A where w is the angular velocity of the rotor in radians per second,
linear plot of tube number against absorption was obtained z is the distance from the rotor center to the boundary, and &/dt
through tube 43. The last fraction had slightly higher extinc- is the velocity of movement of the boundary. The sedimenta-
tion than expected. Presumably this was due to mixing of the tion constant, s, is generally extrapolated to the “standard state”
solutions below and above the level of the needle, resulting from taken as that of water at 20’:
the turbulence produced when the last frothy drops are forced ?m,mbp - Pz0.w)
through the needle. Although it might be expected that the s2om= STm (2)
~ZO,wbp - PT,m)
sucrose concentration would affect the drop size, the volume
difference between the first and last fractions was less than 3%. where fT,rn is the viscosity of the medium at the temperature of
Drops were, therefore, considered to be of constant size, 14.7 f centrifugation, r]20, z. is the viscosity of water at 20”, pp is the
density of the protein in solution (i.e. the reciprocal of the partial
8 Beckman Instruments, Inc., a personal communication. specific volume, i), pT,rn is the density of the medium at the
4 The temperature immediately before and after centrifugation temperature of centrifugation, and ~20,~ is the density of water
was determined in a bucket containing only a sucrose gradient.
6 Subsequent to this investigation, a more elaborate but essen- at 20”. As the partial specific volume of most proteins varies
tially similar fractionator was reported (12). little with temperature (14), p, is generally considered constant.
May 1961 R. G. Martin and B. N. Ames

0.800
3.59 -
3.00 -

0.600

TIME OF CENTRIFUGATION IN HOURS

AT 38,000 RPM
FIG. 2. Theoretical sedimentation behavior of macromolecules
in a 5% (weight per volume) to 20% (weight per volume) sucrose 0.500
012345 6 7 8 9 IO II 12 13 1415 I6 I7
gradient at 3”. The partial specific volumes (fi) in cm3 per g and
sedimentation constants at 20” in water (szo.~) in Svedberg units S20,w’N s
(S) are indicated. FIG. 3. Theoretical curves demonstrating the effect of assumed
martial soecific volume unon the calculation of SZO.+.. These
Equation 2 is applicable to centrifugation in a sucrose gradient, curves ar’e applicable only for the particular sucrose-gradient
employed and for a rotor head of the dimensions of the SW-39.
as it is for centrifugation in uniform media, but in the former case The curves are very steep in the range 0.70 to 0.75 cm3 per g fl.

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both the viscosity (~r,~) and the density (PT,~) are functions
of the sucrose concentration and hence of the distance of the by the enzyme and the time of centrifugation when substances
medium from the rotor center. Combining Equations 1 and 2: of partial specific volume less than 0.80 cm3 per g are examined.
9T.rn Large differences in partial specific volume result in signifi-
ho.wdt = A (pp _ PT,m) * Jx dx (3) cantly different values when the SZO,~ is calculated. If the par-
tial specific volume for a protein is assumed to be 0.800 cm3 per
where the term A = ( pp - p20,,) /qZo, w is a constant for any given g, an s20,ur can be calculated, whereas a slightly different s20,w
partial specific volume (1 /p,) will be arrived at if a partial specific volume of 0.500 cm3 per g
The left-hand side of Equation 3 ran be readily integrated: is assumed. For example, one cannot adequately distinguish
between substances with ~20,~ values of 11.0, 12.0, and 12.8 S
[” S20.,rww
Jo
= sm.&& with corresponding partial specific volumes of 0.500, 0.725, and
0.800 cm3 per g. However, since most proteins have partial
specific volumes between 0.700 and 0.750 cm3 per g (14), the
The right-hand side of Equation 3 can be numerically inte- assumption in all calculations of a partial specific volume of
grated using the trapezoidal approximation: 0.725 cm3 per g will result in less than 3 $& error in the estimation
of s20,ul for most proteins. Alternatively, one may define an
%+I F(x)& = y F(Xi)(Xi+1 - Xi) 0.725
.s~~,~ as the ~20,w calculated on the assumption of a partial specific
s0 0
volume of 0.725 cm3 per g. Correction of the s,“,lz so defined to
+ ; $2 [F(Xi.l) - F(Zi)l[Xi.l - Zil the true s20,u, cannot be accomplished by a simple mathematical
0 ratio if the partial specific volume is determined later. In Fig,
3 the effect of partial specific volume on the calculated s~,,,~ has
This is performed by tabulating arbitrary distances (xi) from been plotted for s~,~~ values of 1 to 15 S. Interpolating from
the rotor center starting at 6.10 cm (the start of the sucrose these curves it is possible to obtain the SQO,,,,of a substance from
gradient) and ending at 9.62 cm (the point at which the sucrose 0’725 when the partial specific volume is available.
its s20,zu
gradient would end if the centrifuge tube were perfectly cylindri-
cal) against sucrose concentration. With standard tables of RESULTS

sucrose molarity against density and viscosity at given tempera- “Xtandard” Enzymes-Three well characterized crystalline en-
tures (2), F(z) = q~,~(zi)/[p, - pr,m(si)]~i can be calculated zymes of different sedimentation rates (yeast alcohol dehydro-
for each distance, zi, and hence the right side of Equation 3 genase, bovine liver catalase, and egg white lysozyme) were
determined for any assumed pp. Plotting zi against & (or zi chosen as standards. In Fig. 4A the sedimentation pattern of
against t for a given w), one obtains a family of theoretical curves catalase after 4 hours of centrifugation at approximately 20” is
for substances of different ~20,~ values and an assumed pp.6 Fig. shown. The dotted line represents the sedimentation pattern
2 shows the theoretical curves for centrifugation at 3” for sub- obtained from a second centrifuge tube run concomitantly in
stances of partial specific volume 0.725 cm3 per g, and ~20,~ values which catalase at the same concentration had been mixed with
of 11.0, 7.4, and 2.15 S. In the same figure are the curves for yeast alcohol dehydrogenase before centrifugation. Fig. 4B
substances of SZO,~ 11.0 S and partial specific volumes 0.500, shows the patterns for yeast alcohol dehydrogenase during the
0.725, and 0.800 cm3 per g. Fig. 2 indicates that a very nearly same experiment.’ In other experiments these two enzymes also
linear relationship should exist between the distance traveled
7 To obtain the greatest reproducibility it was found necessary
0 This method of analysis is similar to that of de Duve et al. to count the number of drops in the last fraction and to plot the
(2) except that a different approximation has been used to find the width of this fraction accordinalv. This has been done in all
numerically determined integral. sedimentation patterns reported: -
1376 Sedimentation Behavior of Enzymes Vol. 236, No. 5

appeared to move at their characteristic rates in pure solution or

combined with other proteins.
Fig. 5 shows an example of the centrifugation patterns of
catalase, yeast alcohol dehydrogenase, and lysozyme obtained
by this method. The dotted lines represent the corresponding
enzymes which have been diluted to the same concentration in a
crude extract of S. typhimurium. The distance in centimeters
from the meniscus to the enzyme peak was estimated to two deci-
mal places by the symmetry of the curve. In Fig. 6 the distances
of the enzyme peaks from the meniscus, as determined from Fig.
5 and many similar centrifugation experiments (all at 3”), are
plotted as a function of the time of centrifugation. The time of
centrifugation was corrected to the equivalent time at 38,000
r.p.m. (The correction to the time of centrifugation at 38,000
r.p.m. includes both the corrections for the actual rotor speed
and for the time of rotor acceleration and deceleration.) A
similar set of curves was obtained at 15”; steeper slopes were FRACTION NUMBER 44 40 36 32 26 24 20 16 I2 6 4
observed.
FIG.5. Lysozyme (0.5 mg), catalase (0.04 mg), and yeast alco-
As predicted, the rate of centrifugation is very nearly constant hol dehydrogenase (0.02 mg) mixed in 0.10 ml of 0.01 M Tris buffer,
for any one of these three enzymes. The .&E” determined for pH 7.5, were layered on a sucrose gradient. After 12.8 hours of

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bovine liver catalase by this method was 11.3 S at 3”. (An centrifugation at 37,700 r.p.m., 3”, the gradient was fractionated
identical value was obtained at 15”.) With the data in Fig. 3 and analyzed (solid lines). In a second gradient, centrifuged at
the same time, these enzymes were diluted to the same final con-
centrations in a crude extract of Salmonella mutant hisG46 (dotted
linfv) .

l =CATALASE
P 0 =ALCOHOL DEHYDROGENASE
A n =RABBIT LIVER SOLUBLE RNA
A=LYSOZYME

TIME OF CENTRIFUGATION
IN HOURS AT 38,OOORPM
6. Each point represents the results of a centrifugation
FIG.
experiment similar to Figs. 5 or 7. All experiments were carried
out at 3”. The time of each experiment has been corrected to the
equivalent time of centrifugation at 38,000 r.p.m. The solid lines
represent the theoretical sedimentation behavior for macromole-
cules of partial specific value 0.725 cm3 per g and the indicated
~20,. values (in Svedberg units).

and if a partial specific volume of 0.73 cm3 per g (15) is assumed,

the calculated s20,w is 11.4 S. Previous investigators (15) re-
DISTANCE FROM I I I I ported a value of 11.3 S (concentration not stated) with optical
MENISCUS IN CM 0.6,52 l.3?5 I.9158 26110 techniques.
FRACTION ’ 40 36 32 26 24 20 16 12
Yeast alcohol dehydrogenase has been reported to have an
NUMBER
s20,2uof 7.2 (1 y. solution) (lo), 7.61 (concentration not stated)
FIG. 4. Sucrose gradients were placed in each of the three (16), and 6.72 S (extrapolated to infinite dilution, centrifugation
buckets of the SW-39 rotor. Catalase (0.04 mg in 0.10 ml) was
at 0’) (17), in the optical centrifuge. An ~20,~ of 7.6 S (concen-
layered on gradient 1, yeast alcohol dehydrogenase (0.02 mg in
0.10 ml) on gradient 2, and a mixture of the two enzymes (0.04 tration, 0.0005%, 25”) has been reported by Kuff et al. with
mg of catalase and 0.02 mg of dehydrogenase in 0.10 ml) on gra- their technique (18). In these experiments an a~,~~ of 7.4 S was
dient 3. The rotor was run at approximately 20” for 4 hours at found. With the reported (17) partial specific volume for this
38,000 r.p.m. A. Catalase activity was assayed in each fraction enzyme (0.769 cm3 per g) and the data of Fig. 3, an ~20,~ of 7.6
of gradient 1 (solid line) and gradient 3 (dotted line). B. Yeast
alcohol dehydrogenase was assayed in each fraction of gradients S was calculated.
2 (solid line) and 3 (dotted line). The .s~~,~ values that have been reported for egg white lyso-
May 1961 R. G. Martin and B. N. Ames 1377

zyme in the optical centrifuge are 2.11 (1.3% protein), 2.09

(0.9% protein), 2.12 (0.5% protein) (19), 1.9 (1.5 to 0.5% pro-
*F Q zt+ tein) (20), 1.8 (concentration not stated) (21), and 1.94 S (1%
UOU
E u .-* protein) (10). Hogeboom and Kuff (lo), using 1% solutions,
obtained s20,w values of 2.01 and 1.88 S. The .&~~ determined
here was 2.1 S; corrected to a partial specific volume of 0.722
(iw+n cm3 per g (19), the SPO,,,,is also 2.1 S.
~~0l.L
owe Soluble RNA, a high molecular weight substance of partial
-I-
specific volume markedly different from protein, was tested on
$3 ;
EOU a sucrose gradient. A typical centrifugation pattern is shown
in Fig. 7. Fig. 6 indicates that soluble RNA also sediments in
DISTANCE FROM o I I I I I this gradient with a constant velocity. The s$lr was calculated
MENISCUS IN CM y 0.65 1.31 1.96 2.61 3.59
to be 4.6 S. The .s~~,~*and partial specific volume of the same
FRACTION 44 40 316 32 :8 24 $0 16 I: 8 4
NUMBER preparation of rabbit liver soluble RNA were determined by
Dr. Samuel Luborsky.9 In a model E Spinco centrifuge
FIG. 7. A centrifugation pattern for rabbit liver soluble RNA
Approximately 0.21 mg of this material in 0.10 ml was layered on a equipped with ultraviolet optics he obtained a pattern with a
sucrose gradient which was 0.2 N in NaCl (no buffer). The frac- single major peak at 4.5 f 0.1 S and small amounts of material
tions were assayed by absorbancy (optical density) at 260 rnr of lower and higher sedimentation rate. The partial specific
after 9.15 hours of centrifugation at 38,000 r.p.m., 3”. volume determined pycnometrically was 0.48 cm3 per g (22).
The so’726
20,,,, of 4.6 S is equivalent to an SZ~,~ of 4.4 S with this par-

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w .080
I I tial specific volume.
Histidine Biosynthetic Enzyme-We have determined the ap-
v) proximate molecular weights of several of the enzymes of histi-
w
= .060 ,”
u dine biosynthesis by examining partially purified enzyme prep-
z
z 5 arations as well as crude extracts. That crude extracts may be
:: .040 2 used for the determination of sedimentation coefficients is dem-
z
z onstrated in Fig. 5. The same sedimentation constants were ob-
Ls
z .020 t- tained with crude and partially purified preparations of the
histidine enzymes.
DISTANCE FROM o t 1 I I
I I Partially purified histidinol dehydrogenase and imidazole
MENISCUS IN CM ? 0.75 I.71 ? 0.65 1.31 acetol phosphate transaminase were placed on sucrose gradients
FRACTION NUMBER 44 40 36 32 28 24 44 40 ;6 32 ;8 24 and centrifuged (Fig. 8). Fig. 9 shows the combined results for
FIG. 8. Sedimentation patterns for histidinol dehydrogenase the centrifugation determinations of crude Nossal extracts and
and imidazoleacetol phosphate transaminase. A crude extract of of the partially purified enzymes. The calculated s$lf values
Salmonella mutant hisEF-135 was centrifuged for 16.50 hours at for these enzymes are 5.1 S for the dehydrogenase and 4.8 S for
33,300 r.p.m., 3”. The dehydrogenase activity is expressed in
change in absorbancy at 600 rnp per minute per 20 ~1 of enzyme the transaminase.
fraction. The transaminase activity is expressed in change in Preliminary studies have been carried out on phosphoribosyl-
absorbancy at 295 rns per 20 minutes per 20 ~1 of enzyme fraction. ATP pyrophosphorylase (22), the first enzyme of the histidine
biosynthetic pathway. This enzyme has a sedimentation con-
I
stant of about 8.6 S.
The centrifugation results with imidazole glycerol phosphate
dehydrase and histidinol phosphate phosphatase indicate aggre-
gation. The sedimentation patterns are complicated in that
multiple peaks appear. Magnesium ions or mercaptoethanol
alters these peaks. A slow moving component is the major one
in the absence of these substances, and there is, primarily, a heavy
component in the presence of mercaptoethanol or magnesium.
The dehydrase and phosphatase activities appeared to migrate
together in several experiments, although further work on this
complicated system is necessary.
15
TIME 0: CENTRI~~GATION
DISCUSSION
IN HOURS AT 38,OOORPM
FIG. 9. Sedimentation behavior of histidinol dehydrogenase The ultracentrifugation technique presented here (2, 3) differs
(0) and imidazoleacetol phosphate transaminase (0). Each from the usual methods of analysis in several aspects. A major
point represents a centrifugation experiment similar to the one attribute of this system derives from the particular sucrose
shown in Fig. 8. The enzyme source for each set of experiments
was a Salmonella his mutant as indicated in the figure. Both gradient employed. The viscosity and density of this sucrose
enzymes were assayed from aliquots of the same set of fractions
obtained after centrifugation. The curves represent the theoreti- * The medium used in Dr. Luborsky’s experiments and our own
cal sedimentation behavior for proteins of partial specific volume was 0.2 M NaCl (no buffer).
0.725 cm3 per g and ~20,~ values of 4.8 S (dotted line) and 5.1 S QS. Luborsky and G. L. Cantoni, to be submitted for publica-
(solid line). tion, Biochim. et Biophys. Acta.
1378 Sedimentation Behavior of Enzymes Vol. 236, No. 5

gradient are such (at least in the temperature range 3” to 15”), TABLE I
that essentially linear migration of most biological materials Molecular weight of hi&dine biosynthetic enzymes
results. Thus, the ratio of the distances traveled from the
There- “Standard”
meniscus by any two substances will always be constant.
fore, if an unknown substance is compared with a standard,
careful control of temperature, time, and speed of centrifugation Lysozyme . . . . .. 63,000 57,000 140,000
are unnecessary. The use of such a standard of known sedi- Alcohol dehydrogenase . .. 86,000 78,000 190,000
mentation coefficient highly simplifies the technique. Experi- Catalase . . .. . ... . 75,000 68,000 160,000
mentally, the ratio, R, can be easily determined after any time Average. 75,000 68,000 170,000
of centrifugation:

R = distance travelled from meniscus by unknown

distance travelled
And because of the nearly
macromolecule :
R=
Or, for macromolecules
from meniscus by standard procedure may be used for enzyme purification. Indeed, small
volumes of enzyme have been partially purified with the SW-39
constant rate of movement of any rotor,lO and good results with much larger volumes have been
achieved in preliminary studies with the SW-25 swinging bucket
rotor.”
~2”;::~of unknown No protein-protein interactions or protein-nucleic acid inter-
s!$,~~ of standard actions were observed in this investigation. Even lysozyme, a
basic protein, showed the same behavior when mixed in a crude
of the same partial specific volume: extract and when pure. This lack of interaction may be due to

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some effect of the sucrose in minimizing interactions or to the
~20.~of unknown
R= (4) fact that very dilute solutions were used compared to what is
~~0.~of standard required in an analytical centrifuge. Nonetheless, protein-pro-
tein interactions are a potential source of error in protein mix-
For substances of similar partial specific volumes, this last equa-
tures.
tion will be very nearly correct. For more accurate determina- The greatest disadvantage of sucrose gradient centrifugation
tions of sedimentation constant, the approach outlined in “The- is the necessity of knowing the partial specific volume in order
oretical” may be followed.
to determine the true s~~,~. However, as discussed previously,
A second aspect which distinguishes this technique from con- the error from the assumption of a partial specific volume of
ventional optical methods is the form of analysis used. Multiple 0.725 cm3 per g for any protein will be small.
fractions of the solution are obtained and these fractions may be A crude estimation of the molecular weight (MIV), can be ob-
analyzed for any of a variety of properties: radioactivity, en- tained from the sedimentation constant alone (13) :
zymatic activity, chemical properties, etc. Thus, a particular
biological material in a multicomponent mixture may be localized 81
-= -MW1 *
by its chemical activity. And hence, the sedimentation coefficient 82 ( MWz J
of a biologically active substance may be determined in a crude
extract. Furthermore, in some cases amounts of material so and for most proteins the ratio sl/sZ is equal to R. (See Equation
small as to be undetectable by the most sensitive optical tech- 4.) This equation derives from the fact that many proteins are
niques may be detectable by another parameter. The disadvan- essentially spherical molecules. Although most globular pro-
tage of any technique in which a parameter other than optical teins, i.e. nearly all enzymes, are only roughly spherical, the
measurement is used is that the centrifugation pattern deter- relationship between s and MW is approximately correct (13).
mined obtains for a particular time of centrifugation, and With the above approximation and the data presented above,
multiple patterns cannot be gotten on the same sample. the molecular weights of the histidine biosynthetic enzymes have
Other systems for the determination of sedimentation coeffi- been calculated (Table I). Lysozyme (MW = 17,200 (19)),
cients in crude extracts, particularly the separation cell of alcohol dehydrogenase (MW = 150,000 (17)), and catalase
Yphantis and Waugh (l), have been demonstrated to be highly (MW = 250,000 (15)) were used as standards.
efficient. In the present system, multiple fractions are obtained The variation in the estimated molecular weight for any partic-
and analyzed, whereas only two are obtained in the separation ular enzyme (Table I) may be due to two factors. The standard
cell. Several advantages arise from the analysis of multiple enzymes vary somewhat in shape, i.e. they are not perfect
fractions: protein aggregation which can easily go undetected in spheres. Also, there is some inaccuracy in the reported molecu-
the separation cell is readily detected; multiple enzymes of lar weights of the standards.
widely divergent sedimentation coefficients can be analyzed in
the same experiment; and high resolution of the sedimentation 10 E. Racker and M. Maver, personal communication.
pattern is possible. In addition, the present technique employs 11One milliliter of a crude extract of Salmonella hisEF 135 was
placed directly on a 29-m& 5 to 40% sucrose gradient and centri-
the less expensive “preparative” ultracentrifuge. A disadvan- fuged for 24 hours in the SW-25 swinging bucket rotor. Thirty
tage of this technique relative to the separation cell is that dif- l-ml fractions were collected. Eighty-seven per cent of the input
fusion coefficients cannot be directly determined. activity of phosphoribosyl-ATP pyrophosphorylase (23) was found
With the use of this technique, a moving zone of material is in four fractions. The two peak tubes containing over 60% of the
input activity had specific activities close to lo-fold greater than
analyzed rather than the boundary of an initially uniform solu- the starting material. A large portion of the nucleic acid present
tion. Because materials of different sedimentation properties in the crude extract was also removed from these fractions, judg-
are separated from each other during the centrifugation, the ing from the absorbancy at 280 and 260 m/L.
May 1961 R. G. Martin and B. N. Ames 1379

We were interested in seeing whether any correlation could be of Hogeboom and Kuff is also applicable to the determination of
made between the molecular weights of the histidine biosynthetic enzymes in protein mixtures and has been used by Levintoe,
enzymes and the genetic complementation data of Hartman et al. Meister, Hogeboom, and Kuff (J. Am. Chem.Sot., 77,5304,1955).
(23, 24). These authors showed that the transaminase gene (C The advantages of zone over boundary determinations have
mutants) has one subunit, the dehydrogenase gene (D mutants) been discussed.
has two subunits, and the pyrophosphorylase gene (G mutants)
has one subunit. These three enzymes have been found to have REFERENCES
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 A Method for Determining the Sedimentation Behavior of Enzymes:
Application to Protein Mixtures
Robert G. Martin and Bruce N. Ames
J. Biol. Chem. 1961, 236:1372-1379.

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