THE JOURNAL OF Bro~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 histidine biosynthesis in Xalmonella typhimurium, it became desirable to determine, in crude extracts, the approximate mo- lecular weights of several enzymes. We have found sucrose gradient centrifugation to be a suitable method for determining sedimentation coefficients of enzymes in protein mixtures. A variety of enzymes of known properties have been studied in the development of the method.

Although the separation cell of Yphantis and Waugh (1) has been demonstrated to be applicable to the determination of molecular weights when multiprotein solutions are used, the present method has several advantages over that system and these advantages will be discussed.

Sucrose gradient centrifugation, using the swinging bucket head of the preparative ultracentrifuge, has been used exten- sively in the determination of sedimentation constants of vi- ruses, mitochondria, microsomes, and ribosomes (2, 3). The adaptation of this method to relatively low molecular weight substances is reported here.

In the sucrose gradient technique the sample to be studied is layered on a gradient and materials of different sedimentation properties separate from each other during the centrifugation. A hole is then punched in the bottom of the centrifuge tube and fractions are collected and analyzed.

With slight modifications in the design of the apparatus for gradient production and fractionation, we have found the method to be simple and accurate. Sedimentation coefficients have been determined for a number of well characterized en- zymes as well as a sample of ribonucleic acid, and the results are in good agreement with the values reported by others. The same values have been obtained whether the enzymes were analyzed as pure proteins or mixed with crude extracts.

Although sedimentation coefficients were directly calculated for a variety of substances in order to determine the accuracy of the method, in general use the procedure may be simplified. The sedimentation coefficient (or approximate molecular weight) of an unknown enzyme may be determined by a simple ratio of mobilities when a standard well characterized enzyme has been added to the protein mixture.

With the use of this technique the sedimentation behavior of several of the enzymes in the pathway of histidine biosynthesis in S. typhimurium has been determined.

* This work was begun during service as an officer in the United States Public Health Service under the Commissioned Officers: Student Training and Extern Program (CO-STEP).

EXPERIMENTAL PROCEDURE

Materials and AssaysLyophilized yeast alcohol dehydro- genase obtained from Worthington Biochemical Corporation was dissolved in 0.05 M Tris-HCl buffer, pH 7.5, to a concentra- tion of 10 mg per ml and stored at 3”. Before use on the sucrose gradient it was diluted to 0.20 mg per ml with the Tris buffer. The dehydrogenase was assayed in a Cary spectrophotometer by following the increase in absorption at 340 rnp for 20 seconds of a l-ml reaction mixture containing 170 pmoles of ethanol, 50 pmoles of Tris, pH 8.5, 15 pmoles of DPN, and 5 to 20 ~1 of enzyme fraction. Units of activity were expressed in terms of change in absorbancy per 20 seconds per 10 ~1 of enzyme frac- tion.

Lyophilized egg white lysozyme (Worthington, twice crystal- lized) was dissolved in 0.05 M Tris buffer, pH 7.5, to a concen- tration of 100 mg per ml and stored at 3”. Before use it was diluted to 5 mg per ml in this buffer. Lysozyme was assayed by following the decrease in turbidity at 650 rnp of a l-ml reac- tion mixture containing 10 pmoles of Tris, pH 8.0, and enough Micrococcus Zysodeikticus cell walls (Difco Laboratories, Bacto- lysozyme substrate) to give an absorbancy of approximately 2.0 absorbancy units in the Cary spectrophotometer. The reaction was started with 5 to 20 ~1 of enzyme fraction and activity was expressed in terms of the change in absorbancy per 20 seconds per 10 ~1 of enzyme fraction.

Beef liver catalase was obtained as an aqueous ammonium sulfate suspension of approximately 40 mg per ml of protein (Worthington). Before use, it was diluted to 0.40 mg per ml in 0.05 M Tris buffer, pH 7.5. Catalase was assayed by follow- ing the decrease in absorbancy at 240 rnp of a 3-ml reaction mixture containing 30 amoles of potassium phosphate buffer at pH 7.5, 18 pmoles of HzOl, and 5 to 20 ~1 of enzyme fraction. Activities were calculated in terms of change in absorbancy per 20 seconds per 5 ~1 of enzyme fraction.

All enzyme assays were approximately linear in the concen- tration ranges used.

Soluble RNA from rabbit liver in a concentration of approxi- mately 6.5 mg per ml (130 absorbancy units per ml at 260 rnp) was kindly supplied by Dr. Samuel Luborsky. The RNA was stored at -15” and diluted with 2 volumes of water before use. It was assayed by its absorption at 260 rnp.

Extracts and Assays of His&line Biosynthetic Enzymes-The histidine mutants were obtained from Dr. P. E. Hartman. The medium, growth of strains, preparation of Nossal extracts, and assays have been previously described (4) with the exception

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

that histidinol was substituted for histidine in the growth media of the histidine-requiring mutants.1 Crude, undialyzed extracts were usually placed directly on the sucrose gradient. In some cases the extract was subjected to Sephadex gel filtration (5) by passing 1 ml of extract through a l- X 5-cm column contain- ing 1 g of Sephadex G-25 (Pharmacia Company, Uppsala, Sweden), equilibrated with 0.01 M Tris, pH 7.5. The protein solution was eluted with the Tris buffer, and the 1.2 ml which contained the protein free from salt were collected.

In order to obtain a partial purification of the histidine en- zymes, a DEAE-cellulose (6) column similar to that previously described was used (7). SaZmonelZa hisG-46 mutant cells were grown and harvested. The cells were suspended in 10 ml of TSM buffer2 and subjected to sonic oscillation for 10 minutes at 0” in a lo-kc Raytheon sonic oscillator. The extract was spun at 25,000 x 9 for 45 minutes and the supernatant was passed through a 3- x 21-cm, 20-g Sephadex G-25 column which had been equilibrated with TSM buffer. The fractions showing appreciable activity were combined. (The specific activity of this sonicated extract was approximately the same as the spe- cific activities obtained on the Nossal extracts,) The enzyme was then purified on a 2- x 25-cm, 8.3-g gravity-packed DEAE- cellulose column, washed according to Peterson and Sober (6), and equilibrated with TSM buffer. The enzyme extract (20 ml) was washed into the column with 5 ml of TSM buffer and then eluted with a linear 0.00 to 0.80 M NaCl gradient in TSM buffer. Fractions of approximately 2.3 ml were collected every 90 seconds. Protein concentrations were determined according to the method of Lowry et al. (8). As previously reported (7), complete separation of histidinol dehydrogenase and imidazole acetol phosphate transaminase from each other and from the histidinol phosphate phosphatase-imidazole glycerol phosphate dehydrase peak was obtained. The ratio of phosphatase activ- ity to dehydrase activity was constant throughout the eluted fractions (7). A IO-fold increase in specific activity was ob- tained for each of the enzymes. The fractions with maximal activity had a 450-fold higher specific activity than wild-type and contained less than 2.5% nucleic acid based on their 280 to 260 rnh ratio of 1.1 (9).

Apparatus for Making Sucrose Gradients-A modification of the simple design of Britten and Roberts (3) was used to pro- duce linear sucrose gradients. Their design consists of a block of Lucite containing two chambers that are connected at the bottom by a removable screw pin. An outflow tube extends from one chamber. This basic design was altered only in that a stopcock was introduced between the two chambers to replace the screw pin. We employed a polyethylene outflow tube which was drawn out in a flame so that emptying time with 2.3 ml of sucrose in each chamber was approximately 10 minutes. The chamber next to the outflow tube was stirred with a platinum bacteriological inoculating loop which was mounted on a motor. The stirring speed was adjusted to give good mixing with mini- mal disturbance of the meniscus.

The apparatus was filled by turning the stopcock to the open position so that free flow existed between the two chambers. The less dense sucrose solution was then added to

1 Specific activities for the histidine enzymes up to 40 times wild type have been observed when Salmonella his mutants are grown on 0.05 rnM histidinol .

* TSM buffer: 0.01 M Tris (free base), 0.005 M magnesium ace- ta,te, and 0.004 M succinic acid, the pH adjusted with NaOH to 7.6,

one chamber and the block rocked back and forth to free air bubbles that might have been caught in the passage between the two chambers. The stopcock was then turned to the closed position and the two chambers were emptied and wiped dry. The block was placed in a clamp, the stirrer adjusted, the out- flow tubing bent upward so that its tip was above the top of the chambers, and each of the two chambers was filled with 2.3 ml of sucrose-buffer solution of the desired concentration. In all the experiments reported here, 20% (weight per volume) of cold sucrose (0.584 M) in 0.05 M Tris-HCl buffer at pH 7.5 was placed in the mixing chamber and 5% (weight per volume) of cold sucrose (0.146 M) in 0.05 M Tris-HCl buffer at pH 7.5 was placed in the adjacent chamber. The rotor was started, the stopcock opened, and the tip of the outflow tube placed at the top of a Lusteroid centrifuge tube. Occasionally gentle suction was required to start the flow. To assure linearity of the gra- dient, care was taken to observe that the fluid levels in the two chambers were equal during emptying.

In initial experiments the production of gradients was tested by mixing dichlorophenolindophenol with the 20 y0 sucrose solution. Perfectly linear plots of absorbancy at 600 mp against fraction number were obtained when 44 fractions were collected. Gradients were stable for at least 48 hours. A 0.05-ml hold-up volume in the apparatus resulted in the delivery of only 4.55 ml to the centrifuge tube.

Layering of Sample-The gradients were stored in a 3” cold room for 4 to 18 hours before use. TO start a run, the substance to be studied was diluted to the desired concentration and 0.10 ml was layered on the gradient; care was taken to avoid bubble formation. It is essential that the material to be layered on the gradient float on 5% sucrose. To insure convection-free sedimentation, protein solutions of considerably less than 5% (approximately 2% or less) must be used (3). A sharp inter- face between the sucrose and protein solution was always ob- served and this interface remained distinct for the several min- utes required to load the centrifuge tubes into the rotor buckets.

Centrifugation-The characteristics and dimensions of the swinging bucket rotor SW-39 designed to fit the model L Spinco centrifuge (Beckman Instruments, Inc., Spinco Division, Palo Alto, California) have been described (lo), as have the errors resulting from the use of nonsector-shaped centrifuge tubes (2). The total volume used in these experiments was 4.65 ml (4.55-ml gradient plus O.l-ml sample) and the distance from rotor center to meniscus, allowing 0.01 cm for rotor stretch and 0.03 cm for radial shift of the meniscus, was calculated to be 6.02 cm (10). Further correction is required because the protein layer is not infinitesimally thin. It was assumed that the protein moves from the middle of the layer. The calculated distance from the rotor center to the middle of the protein layer was 6.06 cm.

Because of the stability rendered to the solution by the sucrose gradient (2, 3) very much less care was needed in the operation of the centrifuge than described by Hogeboom and Kuff (10). The rotor was accelerated very slowly for approximately 10 seconds to eliminate the initial lash given to the rotor by the drive shaft when the two were not fully engaged. After this, 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 the time knob to zero and allowing the rotor to coast to a halt with the brake off.

It is worthy of note that accurate calculations of rotor speed must be based on the odometer readings; the speed setting and

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1374 Sedimentation Behavior of Enzymes Vol. 236, No. 5

tachometer are unreliable.3 During relatively long runs (over 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 rotor to the start of deceleration. Deceleration took 13 minutes from full speed, and integration of the plot of time against speed during deceleration gave an estimated 41 minutes of centrifuga- 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.

Another troublesome aspect of the centrifuge was the main- tenance of a constant temperature during the centrifugation, 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” on our instrument). The precooled head (3”) was then rapidly loaded into the centrifuge. With the above precautions, the temperature increase of the samples during 17 hours of centri- fugation could be kept to less than O.8o.4

Sampling-In their work with CsCl gradient centrifugation, Weigle et al. (11) emptied centrifuge tubes progressively and uniformly by punching a hole in the bottom of each tube with a needle and collecting the drops. We have devised a simple fractronator, based on this principle, which yields a constant number of drops from the 4.65 ml in each tube5 (Fig. 1). In operation, the bottom end piece with its needle was carefully cleaned and forced into place. After the fractionator had been placed in a clamp, the Lusteroid centrifuge tube was carefully lowered into the apparatus with a pair of forceps. The upper end piece was positioned and pressed down, forcing the Lusteroid tube through the needle and starting the flow of drops. The rate of flow could be controlled by the syringe and was kept at approximately 1 drop per second. A soft rubber gasket pre- vented leakage around the needle. With the system described above, it was possible to collect 308 =t 5 drops which were usually divided into 44 fractions (7 drops in each). As the needle rested approximately 0.7 mm above the bottom of the tube, about 3 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 linear plot of tube number against absorption was obtained through tube 43. The last fraction had slightly higher extinc- tion than expected. Presumably this was due to mixing of the solutions below and above the level of the needle, resulting from the turbulence produced when the last frothy drops are forced through the needle. Although it might be expected that the sucrose concentration would affect the drop size, the volume difference between the first and last fractions was less than 3%. Drops were, therefore, considered to be of constant size, 14.7 f

8 Beckman Instruments, Inc., a personal communication. 4 The temperature immediately before and after centrifugation

was determined in a bucket containing only a sucrose gradient. 6 Subsequent to this investigation, a more elaborate but essen-

tially similar fractionator was reported (12).

FRACTIONATOR

THIS END OF END

LUSTEROID TUBE BEING LOWERED INTO POSITION

CUT OFF END OF CENTRIFUGE TUBE

BOTTOM END PIECE

FIG. 1. A Nalgene drying tube with two end pieces (No. 1251 B, Phipps & Bird, Inc., Richmond, Virginia) was cut to 2.5 inches. A Nalgene centrifuge tube, 100 X 16 mm (No. 1210-1, Phipps & Bird, Inc.) with an internal diameter which just allowed easy passage of the Lusteroid centrifuge tube was cut to a 1.5.inch hollow cylinder. This cylinder was forced into the drying tube far enough to allow the bottom end piece of the drying tube to 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 rubber stopper carefully whittled to fit snugly. On this was placed a circular piece of soft rubber with a diameter equal to the internal diameter of the end piece. A 21.gauge syringe needle 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 rubber gasket so that it protruded 1 mm above the latter. A cork with a &mm diameter bore through it was placed in the upper end piece and cut off so that it was even with the plastic ridge of the end piece. The upper end piece was then fitted with a rubber hose to a 50-ml syringe.

0.4 ~1. As an added precaution to maintain drop size, the needle was frequently cleaned with a stylette.

THEORETICAL

The definition of the sedimentation constant ST,,,, in a medium, m, at temperature, T, is given by the equation (2, 13):

where w is the angular velocity of the rotor in radians per second, z is the distance from the rotor center to the boundary, and &/dt

is the velocity of movement of the boundary. The sedimenta- tion constant, s, is generally extrapolated to the “standard state”

taken as that of water at 20’:

?m,mbp - Pz0.w) s2om = STm

~ZO,wbp - PT,m) (2)

where fT,rn is the viscosity of the medium at the temperature of 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 specific volume, i), pT,rn is the density of the medium at the temperature of centrifugation, and ~20,~ is the density of water at 20”. As the partial specific volume of most proteins varies little with temperature (14), p, is generally considered constant.

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

3.59 -

3.00 -

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 gradient at 3”. The partial specific volumes (fi) in cm3 per g and sedimentation constants at 20” in water (szo.~) in Svedberg units (S) are indicated.

Equation 2 is applicable to centrifugation in a sucrose gradient, as it is for centrifugation in uniform media, but in the former case both the viscosity (~r,~) and the density (PT,~) are functions of the sucrose concentration and hence of the distance of the medium from the rotor center. Combining Equations 1 and 2:

9T.rn ho.wdt = A (pp _ PT,m) * J dx (3) x

where the term A = ( pp - p20, ,) /qZo, w is a constant for any given partial specific volume (1 /p,)

The left-hand side of Equation 3 ran be readily integrated:

[” S20.,rww = sm.&& Jo

The right-hand side of Equation 3 can be numerically inte- grated using the trapezoidal approximation:

s %+I F(x)& = y F(Xi)(Xi+1 - Xi)

0 0

+ ; $2 [F(Xi.l) - F(Zi)l[Xi.l - Zil 0

This is performed by tabulating arbitrary distances (xi) from the rotor center starting at 6.10 cm (the start of the sucrose gradient) and ending at 9.62 cm (the point at which the sucrose gradient would end if the centrifuge tube were perfectly cylindri- cal) against sucrose concentration. With standard tables of sucrose molarity against density and viscosity at given tempera- tures (2), F(z) = q~,~(zi)/[p, - pr,m(si)]~i can be calculated for each distance, zi, and hence the right side of Equation 3 determined for any assumed pp. Plotting zi against & (or zi against t for a given w), one obtains a family of theoretical curves for substances of different ~20,~ values and an assumed pp.6 Fig. 2 shows the theoretical curves for centrifugation at 3” for sub- stances of partial specific volume 0.725 cm3 per g, and ~20,~ values of 11.0, 7.4, and 2.15 S. In the same figure are the curves for substances of SZO,~ 11.0 S and partial specific volumes 0.500, 0.725, and 0.800 cm3 per g. Fig. 2 indicates that a very nearly linear relationship should exist between the distance traveled

Large differences in partial specific volume result in signifi- 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 g, an s20,ur can be calculated, whereas a slightly different s20,w will be arrived at if a partial specific volume of 0.500 cm3 per g is assumed. For example, one cannot adequately distinguish between substances with ~20,~ values of 11.0, 12.0, and 12.8 S 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 assumption in all calculations of a partial specific volume of 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 0.725

.s~~,~ as the ~20, w calculated on the assumption of a partial specific volume of 0.725 cm3 per g. Correction of the s,“,lz so defined to the true s20,u, cannot be accomplished by a simple mathematical ratio if the partial specific volume is determined later. In Fig, 3 the effect of partial specific volume on the calculated s~,,,~ has been plotted for s~,~~ values of 1 to 15 S. Interpolating from these curves it is possible to obtain the SQO,,,, of a substance from its s20,zu 0’725 when the partial specific volume is available.

RESULTS

“Xtandard” Enzymes-Three well characterized crystalline en- zymes of different sedimentation rates (yeast alcohol dehydro- genase, bovine liver catalase, and egg white lysozyme) were chosen as standards. In Fig. 4A the sedimentation pattern of catalase after 4 hours of centrifugation at approximately 20” is shown. The dotted line represents the sedimentation pattern obtained from a second centrifuge tube run concomitantly in which catalase at the same concentration had been mixed with yeast alcohol dehydrogenase before centrifugation. Fig. 4B shows the patterns for yeast alcohol dehydrogenase during the same experiment.’ In other experiments these two enzymes also

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: -

0.800

0.600

0.500 012345 6 7 8 9 IO II 12 13 1415 I6 I7

S20,w’N s

FIG. 3. Theoretical curves demonstrating the effect of assumed martial soecific volume unon the calculation of SZO.+.. These curves ar’e applicable only for the particular sucrose-gradient employed and for a rotor head of the dimensions of the SW-39. The curves are very steep in the range 0.70 to 0.75 cm3 per g fl.

by the enzyme and the time of centrifugation when substances of partial specific volume less than 0.80 cm3 per g are examined.

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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 observed.

As predicted, the rate of centrifugation is very nearly constant for any one of these three enzymes. The .&E” determined for bovine liver catalase by this method was 11.3 S at 3”. (An identical value was obtained at 15”.) With the data in Fig. 3

DISTANCE FROM MENISCUS IN CM

FRACTION ’ NUMBER

I I 0.6,52

I I

40 36 32 l.3?5 I.9158 26110 26 24 20 16 12

A

FIG. 4. Sucrose gradients were placed in each of the three buckets of the SW-39 rotor. Catalase (0.04 mg in 0.10 ml) was 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 mg of catalase and 0.02 mg of dehydrogenase in 0.10 ml) on gra- dient 3. The rotor was run at approximately 20” for 4 hours at 38,000 r.p.m. A. Catalase activity was assayed in each fraction of gradient 1 (solid line) and gradient 3 (dotted line). B. Yeast alcohol dehydrogenase was assayed in each fraction of gradients 2 (solid line) and 3 (dotted line).

FRACTION NUMBER 44 40 36 32 26 24 20 16 I2 6 4

FIG. 5. Lysozyme (0.5 mg), catalase (0.04 mg), and yeast alco- hol dehydrogenase (0.02 mg) mixed in 0.10 ml of 0.01 M Tris buffer, pH 7.5, were layered on a sucrose gradient. After 12.8 hours of centrifugation at 37,700 r.p.m., 3”, the gradient was fractionated 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

n =RABBIT LIVER SOLUBLE RNA A=LYSOZYME

TIME OF CENTRIFUGATION IN HOURS AT 38,OOORPM

FIG. 6. Each point represents the results of a centrifugation 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- ported a value of 11.3 S (concentration not stated) with optical techniques.

Yeast alcohol dehydrogenase has been reported to have an s20,2u of 7.2 (1 y. solution) (lo), 7.61 (concentration not stated) (16), and 6.72 S (extrapolated to infinite dilution, centrifugation at 0’) (17), in the optical centrifuge. An ~20,~ of 7.6 S (concen- tration, 0.0005%, 25”) has been reported by Kuff et al. with their technique (18). In these experiments an a~,~~ of 7.4 S was found. With the reported (17) partial specific volume for this enzyme (0.769 cm3 per g) and the data of Fig. 3, an ~20,~ of 7.6 S was calculated.

The .s~~,~ values that have been reported for egg white lyso-

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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+ UOU

tein) (20), 1.8 (concentration not stated) (21), and 1.94 S (1% 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-

$3 ; specific volume markedly different from protein, was tested on

EOU a sucrose gradient. A typical centrifugation pattern is shown

DISTANCE FROM o in Fig. 7. Fig. 6 indicates that soluble RNA also sediments in

I MENISCUS IN CM y

I I I I 0.65 1.31 1.96 2.61 3.59

this gradient with a constant velocity. The s$lr was calculated

FRACTION 44 40 316 32 :8 24 $0 16 I: to be 4.6 S.

8 4 The .s~~,~* and partial specific volume of the same

NUMBER preparation of rabbit liver soluble RNA were determined by

FIG. 7. A centrifugation pattern for rabbit liver soluble RNA Dr. Samuel Luborsky.9 In a model E Spinco centrifuge

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 after 9.15 hours of centrifugation at 38,000 r.p.m., 3”.

of lower and higher sedimentation rate. The partial specific volume determined pycnometrically was 0.48 cm3 per g (22).

I I The so’726 20,,,, of 4.6 S is equivalent to an SZ~,~ of 4.4 S with this par- tial specific volume.

w .080 Histidine Biosynthetic Enzyme-We have determined the ap- v) w ,”

proximate molecular weights of several of the enzymes of histi- = .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 The same sedimentation constants were ob- z Ls

onstrated in Fig. 5. .020 t- tained with crude and partially purified preparations of the

histidine enzymes.

DISTANCE FROM o t I 1 I I I Partially purified histidinol dehydrogenase and imidazole MENISCUS IN CM ? 1.31

FRACTION 0.75 I.71 ? 0.65 acetol phosphate transaminase were placed on sucrose gradients

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 Salmonella mutant hisEF-135 was centrifuged for 16.50 hours at

of the partially purified enzymes. The calculated s$lf values

33,300 r.p.m., 3”. The dehydrogenase activity is expressed in for these enzymes are 5.1 S for the dehydrogenase and 4.8 S for

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

I biosynthetic pathway. This enzyme has a sedimentation con- stant of about 8.6 S.

May 1961 R. G. Martin and B. N. Ames 1377

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

TIME 0: CENTRI~~GATION 15

complicated system is necessary.

IN HOURS AT 38,OOORPM DISCUSSION

FIG. 9. Sedimentation behavior of histidinol dehydrogenase The ultracentrifugation technique presented here (2, 3) differs (0) and imidazoleacetol phosphate transaminase (0). Each point represents a centrifugation experiment similar to the one

from the usual methods of analysis in several aspects. A major

shown in Fig. 8. The enzyme source for each set of experiments attribute of this system derives from the particular sucrose

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 Q S. Luborsky and G. L. Cantoni, to be submitted for publica- (solid line). tion, Biochim. et Biophys. Acta.

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1378 Sedimentation Behavior of Enzymes Vol. 236, No. 5

gradient are such (at least in the temperature range 3” to 15”), that essentially linear migration of most biological materials results. Thus, the ratio of the distances traveled from the meniscus by any two substances will always be constant. There- fore, if an unknown substance is compared with a standard, careful control of temperature, time, and speed of centrifugation are unnecessary. The use of such a standard of known sedi- mentation coefficient highly simplifies the technique. Experi- mentally, the ratio, R, can be easily determined after any time of centrifugation:

R = distance travelled from meniscus by unknown distance travelled from meniscus by standard

And because of the nearly constant rate of movement of any macromolecule :

R= ~2”;::~ of unknown s!$,~~ of standard

Or, for macromolecules of the same partial specific volume:

R= ~20.~ of unknown ~~0.~ of standard (4)

For substances of similar partial specific volumes, this last equa- tion will be very nearly correct. For more accurate determina- tions of sedimentation constant, the approach outlined in “The- oretical” may be followed.

A second aspect which distinguishes this technique from con- ventional optical methods is the form of analysis used. Multiple fractions of the solution are obtained and these fractions may be analyzed for any of a variety of properties: radioactivity, en- zymatic activity, chemical properties, etc. Thus, a particular biological material in a multicomponent mixture may be localized by its chemical activity. And hence, the sedimentation coefficient of a biologically active substance may be determined in a crude extract. Furthermore, in some cases amounts of material so small as to be undetectable by the most sensitive optical tech- niques may be detectable by another parameter. The disadvan- tage of any technique in which a parameter other than optical measurement is used is that the centrifugation pattern deter- mined obtains for a particular time of centrifugation, and multiple patterns cannot be gotten on the same sample.

Other systems for the determination of sedimentation coeffi- cients in crude extracts, particularly the separation cell of Yphantis and Waugh (l), have been demonstrated to be highly efficient. In the present system, multiple fractions are obtained and analyzed, whereas only two are obtained in the separation cell. Several advantages arise from the analysis of multiple fractions: protein aggregation which can easily go undetected in the separation cell is readily detected; multiple enzymes of widely divergent sedimentation coefficients can be analyzed in the same experiment; and high resolution of the sedimentation pattern is possible. In addition, the present technique employs the less expensive “preparative” ultracentrifuge. A disadvan- tage of this technique relative to the separation cell is that dif- fusion coefficients cannot be directly determined.

With the use of this technique, a moving zone of material is analyzed rather than the boundary of an initially uniform solu- tion. Because materials of different sedimentation properties are separated from each other during the centrifugation, the

TABLE I Molecular weight of hi&dine biosynthetic enzymes

“Standard”

Lysozyme . . . . . . 63,000 57,000 140,000 Alcohol dehydrogenase . . . 86,000 78,000 190,000 Catalase . . . . . . . . . 75,000 68,000 160,000

Average. 75,000 68,000 170,000

procedure may be used for enzyme purification. Indeed, small volumes of enzyme have been partially purified with the SW-39 rotor,lO and good results with much larger volumes have been achieved in preliminary studies with the SW-25 swinging bucket rotor.”

No protein-protein interactions or protein-nucleic acid inter- actions were observed in this investigation. Even lysozyme, a basic protein, showed the same behavior when mixed in a crude extract and when pure. This lack of interaction may be due to some effect of the sucrose in minimizing interactions or to the fact that very dilute solutions were used compared to what is required in an analytical centrifuge. Nonetheless, protein-pro- tein interactions are a potential source of error in protein mix- tures.

The greatest disadvantage of sucrose gradient centrifugation is the necessity of knowing the partial specific volume in order to determine the true s~~,~. However, as discussed previously, the error from the assumption of a partial specific volume of 0.725 cm3 per g for any protein will be small.

A crude estimation of the molecular weight (MIV), can be ob- tained from the sedimentation constant alone (13) :

81 MW1 * -= - 82 ( J MWz

and for most proteins the ratio sl/sZ is equal to R. (See Equation 4.) This equation derives from the fact that many proteins are essentially spherical molecules. Although most globular pro- teins, i.e. nearly all enzymes, are only roughly spherical, the relationship between s and MW is approximately correct (13).

With the above approximation and the data presented above, the molecular weights of the histidine biosynthetic enzymes have been calculated (Table I). Lysozyme (MW = 17,200 (19)), alcohol dehydrogenase (MW = 150,000 (17)), and catalase (MW = 250,000 (15)) were used as standards.

The variation in the estimated molecular weight for any partic- ular enzyme (Table I) may be due to two factors. The standard enzymes vary somewhat in shape, i.e. they are not perfect spheres. Also, there is some inaccuracy in the reported molecu- lar weights of the standards.

10 E. Racker and M. Maver, personal communication. 11 One milliliter of a crude extract of Salmonella hisEF 135 was

placed directly on a 29-m& 5 to 40% sucrose gradient and centri- fuged for 24 hours in the SW-25 swinging bucket rotor. Thirty l-ml fractions were collected. Eighty-seven per cent of the input activity of phosphoribosyl-ATP pyrophosphorylase (23) was found in four fractions. The two peak tubes containing over 60% of the input activity had specific activities close to lo-fold greater than the starting material. A large portion of the nucleic acid present in the crude extract was also removed from these fractions, judg- ing from the absorbancy at 280 and 260 m/L.

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May 1961 R. G. Martin and B. N. Ames 1379

We were interested in seeing whether any correlation could be made between the molecular weights of the histidine biosynthetic enzymes and the genetic complementation data of Hartman et al. (23, 24). These authors showed that the transaminase gene (C mutants) has one subunit, the dehydrogenase gene (D mutants) has two subunits, and the pyrophosphorylase gene (G mutants) has one subunit. These three enzymes have been found to have molecular weights of roughly68,000,75,000, and 170,000. Thus, there does not appear to be a correlation between the number of subunits in a gene and the molecular weight of the corresponding enzyme. However, any conclusions based on the molecular weight of an enzyme could be in error if the enzyme is made up of a complex of several monomer units, as is the case for glutamic dehydrogenase (25). Further investigations of genetic map length and of molecular weight are being undertaken and will be necessary before any conclusions concerning a correlation be- tween map length and enzyme size can be made.

We were also interested in trying to determine whether or not the histidine biosynthetic enzymes are associated intracellularly in some sort of functional aggregate. In experiments using su- crose gradient centrifugation, no evidence was found for such an aggregate, even though S. typhimurium extracts were prepared in a variety of ways.

of Hogeboom and Kuff is also applicable to the determination of enzymes in protein mixtures and has been used by Levintoe, Meister, Hogeboom, and Kuff (J. Am. Chem. Sot., 77,5304,1955). The advantages of zone over boundary determinations have been discussed.

REFERENCES

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YPHANTIS, D. A., AND WAUGH, D. F., J. Phys. Chem., 60, 630 (1956).

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BFXTTEN, R. J., AND ROBERTS, R. B., Science, 131, 32 (1960). AMES, B. N., GARRY, B., AND HERZENBERG, L. A., J. Gen.

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SUMMARY

1. Sucrose gradient centrifugation with the swinging bucket rotor of the preparative ultracentrifuge has been used to investi- gate the sedimentation behavior of macromolecules of relatively low molecular weight.

WEIGLE, J., MESELSON, M., AND PAIGEN, K., J. Molec. Biol., 1,379 (1959).

SZYBALSKI, W., Experientia, 16, 164 (1960). SCHACHMAN, H. K., lJltracentri.fugation in biochemistry, Aca-

2. The theoretical characterization of this system is outlined. 3. The applicability of this technique to the determination of

sedimentation constants of enzymes in multicomponent solutions is demonstrated.

demic Press, Inc.’ New York,.19i9. _

EDSALL. J. T.. in H. NEURATH AND K. BAILEY (Editors). The Proteins, Vdl. I, Part B, Academic Press, Inc., New ‘York, 1953.

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SUMNER, J. B., AND GRALI~N, N., J. Biol. Chem., 126,33 (1938). THEORELL, H., AND BONNICHSEN, R., Acta Chem. Stand.. 6,

1105 (1951). HAYES. J. E.. JR.. AND VELICK. S. F.. J. Biol. Chem.. 207. 225

4. Sedimentation coefficients of well characterized macromole- cules were obtained and compared with values determined by optical techniques. Good agreement was observed.

5. The general applicability of this system to the determina- tion of sedimentation constants and enzyme purification is dis- cussed.

(1954). ’ ’ I , , ,

HUFF, E. L., HOGEBOOM, G. H., AND STRIEBICH, M. J., J. Biol. Chem., 212,439 (1955).

WETTER, L. R.. AND DEUTSCH, H. F., J. Biol. Chem., 192, 237 (1951):

ALDERTON, G., WARD, W. H., AND FEVOLD, H. L., J. Biol. Chem., 167, 43 (1945).

6. Sedimentation coefficients were determined for three histi- dine biosynthetic enzymes.

21. ABRAHAM, E. P., Biochem. J., 33, 622 (1939). 22. AMES, B. N., MARTIN, R. G. AND GARRY, B., J. BioZ. Chem.,

in press. 7. Some observations on the correlation of genetic information

with enzyme molecular weight are discussed.

23. HARTMAN, P. E., LOPER, J. C. AND HERMAN, D., J. Gen. Micro- biol., 22, 323 (1960).

24. HARTMAN, P. E., HARTMAN, Z., AND HERMAN, D., J Gan. Microbial., 22, 354 (1960).

Addendum-We wish to emphasize that the boundary method 25. FRIEDEN, C., J. BioZ. Chem., 234, 899 (1959).

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Robert G. Martin and Bruce N. AmesApplication to Protein Mixtures

A Method for Determining the Sedimentation Behavior of Enzymes:

1961, 236:1372-1379.J. Biol. Chem. 

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