AN ELECTROPHORETIC STUDY OF THE SALT FRACTIONATION OF YEAST EXTRACTS*

BY KURT G. STERN, ARNOLD H. SCHEIN, AND JAMES S. WALLERSTEIN

(From the Department of Chemistry, Polytechnic Institute, Brooklyn, the Department of Physiology and Biochemistry, New York Medical College, and the Overly

Biochemical Research Foundation, New York)

(Received for publication, May 8, 1946)

The present experiments were undertaken after completion of the elec- trophoretic analysis of crude maceration extracts from dried top and bottom yeasts, which formed the subject of an earlier publication (1). It was found that extracts of this type, commonly designated as Lebedev juice, contain a number of electrochemically different colloidal components. Upon charting the “molecular spectrum” with the aid of the Tiselius elec- trophoresis apparatus, marked quantitative differences were found to exist in the diagrams obtained with the extracts from various yeast types. Furthermore, the electrophoretic patterns of undialyzed maceration ex- tracts appear to be more complex than those of the dialyzed solutions.

Yeast maceration extracts are known to contain a large number of sub- stances, both of protein and non-protein character. Thus, in addition to the complete enzyme system of alcoholic fermentation (zymase complex), hydrolytic enzymes, dehydrogenases, flavoproteins, and hemoproteins (cj. (2)), as well as “inert” soluble proteins and polysaccharide (yeast gum), have been demonstrated in such preparations. Perhaps the most widely employed technique for isolating biologically active proteins from such sources as yeast or tissue extracts is the fractional salting-out method in which are utilized the differences in solubility of individual proteins in solu- tions of ammonium sulfate and other neutral salts of varying degrees of saturation. Frequently the salting-out method is combined with iso- electric precipitation procedures or with a fractionation involving the use of water-miscible organic solvents, especially alcohol and acetone. Al- though ultracentrifugal and electrophoretic techniques of separating bi- ological colloid mixtures possess, as a rule, a higher degree of specificity and resolving power than chemical fractionation methods, the latter still remain the only ones practical on a larger scale owing to the limited capacity of the physical tools mentioned above. Their chief application at the

* The experimental part of this work was completed at the Overly Biochemical Research Foundation in 1944. It formed part of a research project conducted under the auspices of the Food Distribution Administration, United St.ates Department of Agriculture.

59

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60 ELECTROPHORESIS OF YEAST FRACTIONS

present time consists in their use in controlling the results of chemical fractionation procedures. For this reason it was thought of interest to follow the ammonium sulfate fractionation of bottom yeast maceration extracts by electrophoretic analysis of the system at various stages of the process. This affords a correlation of chemical and electrochemical data of a type which has been found useful in the field of serum and tissue proteins (cf. (3)).

EXPERIMENTAL

Materials and Preparation of Yeast Extracts-10 pound batches of washed and pressed brewers’ bottom yeast were obtained through the courtesy of the Krueger Brewing Company of Newark, New Jersey. The yeast was driven through a coarse sieve and then dried in layers of about 1 cm. in height over a period of several days at 30”. It has been established that the cell membranes are ruptured by partial autolysis under these conditions. This explains why aqueous maceration extracts from such dried yeast prepa- rations contain the enzyme system of alcoholic fermentation (zymase) in soluble and usually active form, in contrast to similar extracts obtained from fresh or from more rapidly dried yeast. The dried yeast was stored at room temperature. At the time when the present experiments were performed, the dried yeast preparations were 6 to 8 months old. Lebedev juice is usually prepared from dried yeast by maceration with tap water at 37”. In the present work, neutral phosphate buffer was substituted for the water, in accordance with the recent experiences of other investigators (4) who found that the presence of phosphate ions is beneficial for the enzymatic and fermentative activity of yeast extracts. Furthermore, the method of salt fractionation here adopted was originally carried out on such phosphate extracts from yeast (see below).

Ammonium Sulfate Fractionation-The relative paucity of information on the fundamental properties of the proteins present in yeast extracts made the choice of a specific fractionation procedure somewhat difficult. It was finally decided to follow the method of ammonium sulfate fractiona- tion employed by Green, Herbert, and Subrahmanyan (5) in their attempt at isolating yeast carboxylase. Ammonium sulfate has previously been used as a stabilizing and extracting agent for this enzyme (6). Inasmuch as the aim of the present investigation was the electrophoretic analysis of the various components obtainable from yeast extracts by salt fractionation rather than the isolation, in pure form, of a specific constituent, such as carboxylase, all fractions were examined in the Tiselius apparatus without regard to their enzymatic activity.

Electrophoresis Technique-The technique of electrophoretic analysis of the various extracts and fractions obtained in this study and the Tiselius

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STFXX, SCHEIPI;, AND WALLERSTEIN 61

apparatus employed were the same as in the preceding report (1). Inas- much as it was desirable to establish the experimental conditions most likely to yield reproducible results, all solutions were equilibrated against the supernatant buffer by dialysis through cellophane at low temperature, even though this might cause a loss of enzymatic activity through the diffusion of coenzymes and low molecular activators. The buffer chosen for the majority of the electrophoresis experimenh was prepared by mixing 94 part.s of secondary and 6 parts of primary sodium phosphate; the molar- ity with regard to total phosphate was 0.05, the ionic strength was approx- imately 0.12, and the pH close to 7.3. In order to ascertain that the protein solut.ions had been properly equilibrated against. the buffer, the conductivity as well as the hydrion concentration of all solutions was determined after dialysis. The non-dia.lyzable, presumably protein nitrogen of the solutions studied varied from 2.3 to 0.15 mg. per ml. It decreased with the progress of t.he fractionation, although the ammonium sulfate precipitates were re- dissolved for examination in progressively smaller volumes of solvent.

The potential gradients employed in the electrophoresis experiments ranged from F = 2.6 t.o 3.6 volts per cm. The ground surfaces of the electrophoresis cell were lubricated with the Vaseline-paraffin oil mixture recommended by Tiselius instead of the Celloscal grease employed in the previous experiments (1). The diagrams were recorded at a photographic magnification factor of 1.074l and were further enlarged 3.45 times by projection for tracing purposes and subsequent planimetry.

The solutions studied in this work were, as a rule, clear; they were either colorless or yellow. In order to prevent a shading of the plates due to the color which tends to be accentuated by the spectral distribution of the light source employed (General Electric mercury high pressure burner, type H-4), Wratten panchromatic plates, or Eastman trichromatic X- plates, type B, were used for the recording of the electrophoresis patterns by Longsworth’s schlieren scanning technique (7).

Observations and Results

In the course of this work a total of three fractionations was carried out, according to the scheme shown in the accompanying flow sheet.

Inasmuch as all fractionations were performed in a similar manner, only one of them, viz. Preparation 2, will be described in some detail.

Slep 1-500 gm. of dried Krueger’s bottom yeast, Batch 12a, were sus- pended in 1500 ml. of 0.066 M phosphate buffer of pH 7.2. The suspension

1 The mobility data reported in the preceding paper (1) were computed on the basis of a photographic magnificnt.ion factor of 1.37. Inasmuch as a recalibration of the apparatus revealed that the actual value is 1.074, the mobilities quoted in the previous publication were too high by a factor of 1.2i.

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62 ELECTROPHORESIS OF YEAST FRACTIONS

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STERN, SCHEIN, AND W.iLLERSTEIN

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64 ELECTROPHORESIS OF YEAST FRACTIONS

was stirred mechanically for 1 hour at 36-37” on the water bath. 2000 ml. of tap water of room temperature were added and the diluted suspension was placed for 1 hour in the refrigerator. It was then centrifuged for 20 minutes at 2000 R.P.M. The supernatant Lebedev juice (Fraction I) which was decanted from the sediment had a volume of 2570 ml. and a nitrogen content, as determined by I’rcgl’s micro modification of the Kjeldahl method, of 2.3 mg. per ml. ,4n aliquot of 20 ml. was dialyzed in the refrigcrat.or against 2 liters of 0.05 M phosphate buffer, pH 7.7, and examined in the electrophoresis apparatus. Tht! diagram obtained in this way (Esperimcnt. 317) is reproduced in Fig. 1. It discloses the presence of five more or less resolved components of a mobility ranging from -0.74 to -6.0 X 10e5 cm.2 per second per volt for the descending boundaries. The major amount of material was cont.ained in t,he fraction of intermediate mobility (see Table I). The pattern here obtained resembles that yielded by a similar estract in the previous publication ((I), Fig. 9), except that

FIG. 1. IClectrophoretic diagram of crude, dinl~artl l~hctlev juice from Krueger’s bottom yeast (13utch 12s), Iiraction 1.

the resolution of the intermediate fraction is greater in the present instance. The nomenclature employed in the present paper with regard to the desig- nation of the various elcctrophorctic components is the same as that used previously (1); w do not wish to imply, however, that components bearing the same dcsignstion arc ncccssarily identical. It is quite possible that the quasistationary peaks (designated as y component) are in part buffer and protein concentration gradients (boundary anomalies), similar to the 6 and c maxima obscrvcd in the electrophoretic diagram of blood serum (cj. (8)).

Step II--The remainder of the Lebedev juice (2550 ml.) was mixed with 400 ml. of 0.5 M phosphate buffer, pII 7.0, and then with 200 ml. of M

calcium acetate solution. The heavy precipitate, consisting largely of calcium phosphate, was centrifuged off (10 minutes at 2000 R.P.M.) and discarded. The volume of the clear, yellow supernatant solution (Frac- tion II) amounted to 2810 ml.; the nitrogen content was 2.06 mg. per ml., indicating that little if any protein had been adsorbed on the calcium phosphate. Of this solution, 20 ml. were equilibrated against 0.05 M

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STEBN, SCHEIN, AND WALLERSTEIN 65

phosphate buffer, pH 7.7, prior to examination in the Tiselius apparatus. The pattern recorded (Experiment 316) was very similar to that obtained from the original Lebedev juice (see Fig. 1). The differences in apparent mobility of the various components (see Table I) compared with those present in the Lebedev juice are possibly due to a slight hydrostatic shift which may have occurred during the experiment in the Tiselius cell (note the apparent cathodic mobility of the y-boundary).

Step III--To the balance of the supernatant fluid remaining after the calcium phosphate precipitation (2790 ml.) there were added 964 gm. of purified ammonium sulfate to bring the solution to 0.5 saturation. A pre- cipit,ate formed immediately upon addition of the salt. The suspension was kept in the refrigerator overnight and was then filtered by suction on a large Btichner funnel through filter paper covered with a 3 mm. layer of

FIG. 2. Electrophoretic diagram of Fraction IIIb

Hyflo Super-Cel. The clear, yellow filtrate (Fraction IIIb), upon ex- amination (Experiment 318) in the Tiselius apparatus after dialysis against phosphate buffer, showed a composition somewhat resembling that of the preceding solutions but the peaks visible in the electrophoretic diagram (Fig. 2) enclose appreciably smaller areas, in keeping with the lower total nitrogen content (0.71 mg. of N per ml.). The rest of the filtrate was discarded. The filter cake remaining after removing as much of the filtrate as possible by pressing was suspended in 750 ml. of 0.04 M citrate buffer of pH 6.0. A slight turbidity was removed by filtration and an aliquot of the clear, yellow solution (Fraction IIIa) was subjected to electro- phoretic analysis (Er periment 319). The resulting diagram (Fig. 3) showed the presence of three to four components with mobilities ranging from -0.1 to -6.27 X 10T5 cm.2 per second per volt (Table I). The nitrogen content of the solution was 1.32 mg. per ml.

Step IV-The remainder of the solution was brought to 0.5 saturation by adding 305 gm. of ammonium sulfate to approximately 800 ml. of the solution. After storage overnight in the refrigerator, the suspension was filtered by suction through Hyflo Super-Cel. The clear, greenish yellow filtrate (Fraction IVb), which contained only a trace of protein, was dis-

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66 ELECTHOPHOHESIS OF YEAST FRACTIONS

carded. The precipitate remaining on the filter was dissolved in 500 ml. of 0.04 Y cit.rat.e buffer, pH 6. A small amount of insoluble material was removed by Wration and an aliquot of the clear filt.rate (Fraction IVa) was esamined in the Tiselius apparatus (Experiment 320). The resulting pattern was very similar to that recorded with Fraction ITIa (see Fig. 3), as would be expected from the fact that most of the protein material had been recovered in this fraction. Fraction IVa contained 1.56 mg. of N per ml. or 87 per cent. of the total nitrogen present in Fraction IIIa.

Xlep I’--The material, contained in Fraction IVa, representing proteins precipitable at 0.5 saturation with ammonium sulfat.e, n-as now further fractionated as follows: To 530 ml. of solution of $‘raction 11-a were added 185 ml. of saturated ammonium sulfate solution, yielding a 0.26 saturated solution. ;\fter standing in the cold overnight, the suspension was filtered

-+- DESCENDING ASCENDING----+

PIG. 3. IGctrophoretic diagram of lJr:tctiorl III:*

through Hyflo Super-Gel. The precipitate remaining on the filter was dissolved in 100 ml. of 0.05 M phosphate buffer, pII 7.7, and filtered to obtain a clear solution. According to clectrophoretic analysis (Experi- ment 321, Fig. 1) this fraction, designated as SlJ?fraction 1, contained two major components (CY and /3) of a mobility of -6 and -i X 10e5 cm.* per second per volt respectively. The descending patterns revealed, in addi- tion, the prcscnce of a trace of a highly mobile material (u = -8.7). The nitrogen content amounted to 0.4 mg. per ml. To the filtrate re- maining after precipitating Subfraction 1 at 0.26 saturation with ammonium sulfate there were added 350 ml. of saturated ammonium sulfate solution, raising the salt concentration to 0.5 saturation. A bulky precipitate formed which was filtered off as usual and dissolved in 300 ml. of 0.05 M phosphate buffer of pH 7.7. After clarification, an aliquot was examined in the Tiselius apparatus (Experiment 322) and found to contain essen-

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STERN, SCHEIN, AND W.4LLERSTEIN 67

tially material of intermediate mobility (a components), as shown in Fig. 5 and Table I.

The solution contained 1.M mg. of S per ml.; it, was designated Sub- fraction 2. The filtrate remaining after t,he removal of Subfraction 2 (volume 990 ml.) was brought to 0.73 saturation by the addition of 870 ml. of saturated ammonium sulfate solution. The resulting precipitate was dissolved in a mixture of 50 ml. of 0.1 hc citrate buffer, pH 6, 50 ml. of

,+---DESCENDING ASCENDING--+

FIG. 4. Electrophoretic diagram of Subfraction 1

i +--DESCENDING ASCENDING- FIG. 5. Electrophoretic diagram of Subfraction 2

saturated ammonium sulfate solution, and 100 ml. of distilled water, cor- responding to 0.25 saturation with regard to ammonium sulfate. An sliquot of the filtered solution, called Subjradm S, was subjected to electrophoretic study (Experiment 323). The diagram (Fig. 6) showed a relatively large amount of an initially well defined fraction of a mean mobility of -4.17 X 1O-6 cm.2 per second per volt and a smaller amount of a component of a mean mobility of -6.3. As in the instance of most other components here observed, the former showed a considerable tendency to spread in the later part of the experiment. The nitrogen content of the solution was 0.49 mg. per ml. The filtrate obtained after precipitating

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68 ELECTROI’HORESIS OF YEAST FRACTIOKS

Subfraction S gaye a negative test for protein with sulfosalicylic acid and hence was discarded. The nitrogen content of all three subfractions amounted to 73 per cent of t.he nitrogen content of Fraction IVn; the bulk of the nitrogen, viz. 57 per cent, was contained in Subfraction 2, while Subfraction 1 cont.ained about 5 per cent and Subfraction S represented approximately II per cent of the nitrogen of Fraction IVa.

Step VI-Subjradion S, which, according to Green et al. (5), would be expected to contain the bulk of the yeast carboxylase, was further frac- tionat.ed in the following manner. The remainder of Subfraction 3,

A A

8 B

c

D c

ASCENDING BOUNDARY F--+

Fin. 6. I~lectrophoretic diagram of T:IG. 7. Elect.roplioretic tli:tgr:tm of Subfraction 3. Subfraction 311.

amounting to 180 ml., was first brought to 0.5 saturation of ammonium sulfate by the addition of 80 ml. of saturated salt solut.ion. Since no precipitate, corresponding to Green’s Subfraction Sa, formed at this stage, an additional 143 ml. of saturated ammonium sulfate solution were added, raising the salt concent,ration to 0.67 saturation. The resuhing precipitate was dissolved in 40 ml. of 0.1 M citrate buffer, pH 6, 40 ml. of saturated ammonium sulfate solution, and 80 ml. of water. Of this solution, repre- senting Subfraction Sb, 15 ml. were dialyzed against phosphate buffer and submitted to electrophoretic examination (Experiment 324). The diagram (Fig. 7) showed essentially one, very sharp boundary migrating at pH 7.32 at a mobility of -5.7 X 10m5 cm.2 per second per volt in the ascending and -6.0 in the descending limb of the apparatus. This boundary, which showed only a slight tendency to spread during electrophortis, would correspond to an (Y component on the basis of its mobility (see Table I).

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STERN, SCHEIN, AND WALLERSTEIN 69

The filtrate obtained after the removal of Subfraction 35 was brought to 0.82 saturation by the addition of 292 ml. of saturated ammonium sulfate solution to 380 ml. of protein solution. The precipitate which now formed was dissolved in 20 ml. of 0.05 M phosphate buffer, pH 7.7, clarified by filtration @+&fraction SC), and studied in the Tiselius apparatus (Ex- periment 325). According to the pattern recorded of this fraction (Fig. 8) several components of mobilities ranging from -3.0 to -7.1 X 10” cm.2 per second per volt (for the ascending boundaries) were present in this subfraction. 82 per cent of the material was of p mobility (see Table I). All boundaries showed a considerable tendency to spread during electro- phoresis, in contrast to the boundary observed in Subfraction Sb. The nitrogen content of Xubfmction Sc was 0.5 mg. per ml.

+---DESCEN~ING ASCENDING-

FIG. 8. Electrophoretic diagram of Subfraction 3c

Step VII-Subfraction Sb, which according to Green contains the enzyme carboxylase, was further fractionated as follows: To 140 ml. of Subfrac- tion Sb there were added 57.5 ml. of saturated ammonium sulfate solution, bringing the solution to about 0.5 saturation. Since no precipitate, corre- sponding to Green’s Subfraction Sbl, appeared at this point, 115 ml. of saturated ammonium sulfate solution mere added, raising the concentration to 0.66 saturation. The precipitate which formed was dissolved in 30 ml. of 0.04 M citrate buffer, pH 6; the solution was clarified by filtration (Sub- fraction 3b2). One-half of this solution was first used for electrophoretic analysis (Fig. 9, Experiment 326). Only one boundary of a mobility of -5.4 X 10-s cm.2 per second per volt was recorded on the ascending as well as the descending side (see Fig. 10). This boundary, which exhibited the mobility of an a-protein fraction, showed a considerably greater tendency to spread during electrophoresis than did the single boundary observed in Subfraction Sb (compare with Fig. 7). The nitrogen content of this solution was less tha.n 0.68 mg. per ml. The material was recovered from the electrophoresis cell, combined with the remainder of Subfraction 3b2, and was precipitated at full saturation with ammonium sulfate. The

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70 ELECTROPHORESIS OF YEAST FRACTIONS

precipitate was redissolved in 7 ml. of 0.05 M phosphate buffer, pH 7.7, and the resultant clear, yellow solution was equilibrated against 2 liters of phosphate buffer by dialysis. When this reconcentrated Subfraction Sbz was examined in the Tiselius apparatus, the appearance of the (Fig. 10, Experiment 329) electrophoretic pattern in the ascending limb was the same as that of the original Subfraction Sbs, except for the presence of a

f-- DESCENDING ASCENDING.W

FIG. 9. Electrophoretic diagram of Subfraction 3bz

ASCENDING I---+

ASCENDING BouNDARV B~uN~AFw - 1

FIG. 10 FIG. 11

FIG. 10. Electrophoretic diagram of Subfraction 3b,, concentrated by ammonium sulfate precipitation.

FIG. 11. Electrophoretic diagram bf Subfraction 3ba.

trace of a component of lower mobility. The mobility of the main com- ponent amounted to -4.4 X 10e5 cm.2 per second per volt, which is some- what lower than that of the corresponding material in the original sub- fraction. This might indicate a modification of the protein under the influence of high salt concentration.

The filtrate from Subfraction 3b2 was brought to 0.83 saturation by adding 330 ml. of saturated ammonium sulfate solution to 330 ml. of the filtrate. The precipitate which formed was dissolved in 11 ml. of 0.05 M phosphate

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STERN, SCHEIN, AND WALLERSTEIN 71

buffer, pH 7.7, clarified by filtration (Subjm.&m 3bs), and subjected to electrophoretic examination a.fter equilibration against the same buffer (Experiment 327, Fig. 11). As would be expected from the low nitrogen content (0.15 mg. per ml.), only a very low .peak was observed in this subfraction (Fig. 11). The mobility of this material, viz. -3.6 X 104 cm.” per second per volt, was definitely lower than that of the boundary recorded for Subfraction Sbn. The mobility values as well as estimates of the relative concentrations of the various electrophoretic components observed in these experiments are given in Table I.

xo claim is made with regard to the precision of these data. They are listed chiefly as an aid in labeling and tracing the individual components.

DISCUSSION

The optical resolution of crude dialyzed yeast maceration extracts into individual components by the moving boundary method of electrophoresis is considerably less complete than that of the proteins in blood serum, for example. Under these conditions a successful mechanical separation of the individual colloids present in these extracts by preparative electrophoresis would not seem to be very promising. However, the present experiments show that electrophoretic analysis is useful in determining the success of chemical fractionation procedures at every step of the process. As judged by the results obtained with this physical tool, half saturation of crude yeast extracts with ammonium sulfate does not produce a fractionation as decisive as that of blood serum into globulin and albumin components by the same.means. Especially pronounced in the case of yeast proteins is the tendency -of coprecipitation of components of high and intermediate ammonium sulfate solubility in the early stages of the fractionation pro- cedure. From the point of view of extent of separation, Step II (calcium phosphate precipitation) appears to be the least efficient and Step IV (fractional ammonium sulfate precipitation) the most effective operation. It is also noteworthy that fractions of similar electrophoretic mobility, present in the crude extracts, exhibit significant differences with respect to their solubility in ammonium sulfate. This is similar to the finding of Tiselius (3) that the individual globulin components of blood serum con- tain fractions of different solubility in spite of closely similar electro- phoretic mobility.

As may be seen from the diagrams reproduced in this paper, the pro- cedure of Green et al., employed in the purification of yeast carboxylase (5), leads to the isolation of a protein component which has a well defined mobility (5.4 X 10” unit at pH 7.3) and a fair degree of electrochemical homogeneity. It should be noted, however, that Green et al. state that their enzyme preparation of the highest activity ratio which they were

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ES

peri-

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No

.

317

316

319

318

320

321

322

323

TABL

E I

Elec

lroph

oret

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alys

is

of Y

east

Mac

erat

ion

Eztra

ct

(Kru

eger

’s Bo

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6)

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Pr

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by A

mm

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te

Prec

ipita

tion

Frac

tion

Lebe

dev

juice

Filtr

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um

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t.,

0.50

am

mon

ium

su

lfate

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tura

tion,

di

ssol

ved

and

repr

ecip

itate

d Fi

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m

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sat

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amm

oniu

m

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te

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n,

disc

arde

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t.,

0.50

am

mon

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su

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56

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84

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96

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325

326

327

329

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74 ELECTROPHORESIS OF YEAST FRACTIONS

able to obtain was not homogeneous. As is shown in this paper (see Figs. 9 and lo), the reprecipitation by ammonium sulfate of the protein isolated by Green’s method produces a change in electrophoretic behavior and pattern. Upon examination in the analytical ultracentrifuge this ma- terial was found to be polydisperse. It would appear, therefore, that in highly purified form this protein shows a greater sensitivity towards high salt concentration than in the crude extract, an experience not uncommon in the field of biologically active proteins. It would be of interest to correlate the electrophoretic components here observed with proteins previ- ously isolated from yeast; e.g., the crystalline proteins described recently by Kunitz and McDonald (9).

SUMMARY

The fractionation of the proteins present in yeast maceration extract (Lebedev juice), essentially by ammonium sulfate precipitation, has been followed by electrophoretic analysis of the various fractions in the Tiselius apparatus.

It could be demonstrated that the procedure employed by Green and his associates in the purification of yeast carbaxylase leads to the isolation of a fairly well characterized protein fraction. This protein occurs in the crude extracts only in small amounts and it has been possible to follow the elimination of large amounts of ballast proteins and other colloids by controlling the chemical fractionation procedures by electrophoretic analy- sis at each stage of the process. No attempt has been made to correlate specific biological activity with the electrochemical properties of the individual components. Data are presented for the electrophoretic mobili- ties and approximate relative concentrations of the various colloidal com- ponents of yeast maceration extract.

BIBLIOGRAPHY

1. Stern, K. G., J. Biol. Chem., 162, 345 (1944). 2. Oppenheimer, C., and Stern, K. G., Biological oxidation, The Hague (1939). 3. Tiselius, A., Biochem. J., 31, 1464 (1937). 4. Neuberg, C., and Lustig, H., qrch. Biochem., 1, 191 (1942). 5. Green, D. E., Herbert, D., and Subrahmanyan, V., J. Biol. Chem., 138,327 (1941). 6. Melnick, J. L., and Stern, K. G., Enzymologia, 8, 129 (1940). 7. Longsworth, L. G., J. Am. Chem. Xoc., 81, 529 (1939). 8. Tiselius, A., and Kabat, E. A., J. Exp. Med., 83,119 (p. 126) (1939). 9. Kunite, M., and McDonald, M. R., J. Gen. Physiol., 29, 143 (1946).

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WallersteinKurt G. Stern, Arnold H. Schein and James S.

YEAST EXTRACTSTHE SALT FRACTIONATION OF

AN ELECTROPHORETIC STUDY OF

1946, 166:59-74.J. Biol. Chem. 

  http://www.jbc.org/content/166/1/59.citation

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