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ULTRACENTRIFUGAL ANALYSIS OF PEPSIN-TREATED SERUM GLOBULINS1~2 MARY L. PETERMANN
Department of Chemistry, Universaly of Wisconsin, Madison, Wisconsin Received J u l y 18, 1941 I. INTRODUCTION
Recently it has been found possible to purify antibodies by digesting immune sera with proteolytic enzymes a t hydrogen-ion concentrations well removed from the optimum for enzyme action. While some digestion into small fragments takes place, the antibody activity is not destroyed, and the ratio of antibody activity to protein concentration is considerably increased (3, 5 , 10). The antibody is, however, split into large fragments, some of which carry the activity. In this laboratory the sedimentation behavior of such fragments has already been studied for horse diphtheria antitoxin (6) and for horse antipneumococcus antibody (7, 8). Also, in a preliminary communication Northrop (4)has mentioned a sedimentation determination on diphtheria antitoxin digested with trypsin. Most studies of proteolysis, on the other hand, have been concerned only with the amount of non-protein material formed. However, some experiments have been performed on the digestion of egg albumin by papain (1) and by pepsin (12). Both the large and the small fragments formed in digestion were investigated by physical methods. This study of the intermediate products of the action of pepsin on beef globulins was therefore undertaken with several purposes in mind. These were, first, to acquire information which might be useful in antibody digestions; second, to compare the size and relative amount of fragments obtained by splitting pseudoglobulins and euglobulins; and third, to investigate peptic digestion a t hydrogen-ion concentrations between those used in antibody purification and the optimum for peptic digestion. 11. METHODS
A . Preparation of globulins One volume of cold beef serum was diluted with an equal volume of cold water. The globulins were precipitated by the slow addition of two volumes of saturated ammonium sulfate. The chilled solution was stirred continuously during the addition of the salt. After the system had stood in a refrigerator for from 12 to 18 hr., the supernatant liquid was decanted off and the precipitate packed in a Presented at the Eighteenth Colloid Symposium, which was held at Cornel1 University, Ithaca, New York, June 19-21, 1941. * The expenses of this investigation were defrayed by the Wisconsin Alumni Research Foundation. Assistance in the construction of accessory apparatus was furnished by the personnel of Work Projects Administration Official Project No, 65-1-53-2349.
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laboratory centrifuge. The precipitated globulins were washed twice with cold, half-saturated ammonium sulfate, dissolved in cold water, and dialyzed against cold water until the precipitate had settled out and the supernatant was clear. The whole precipitate constituted the “euglobulin” and the supernatant contained the “pseudoglobulin” used in these experiments. No further attempts at fractionation were made, although it was realized that both globulins were undoubtedly mixtures.
B . Digestion procedure Two digestion procedures were employed. The first was that used by Pope (10) and the author (6) for the digestion of horse diphtheria antitoxin. A 1 per cent solution of the protein containing 5 per cent of sodium chloride was brought to pH 4.2 ( g b s electrode) by the addition of solid citric acid. Four milligrams of crystalline pepsin per 10 cc. of solution waa then added. After standing at room temperature for 30 min., the solution waa heated at 58°C. for 45 min. The precipitate was removed by centrifugation, and the supernatant dialyzed against a phosphate-borate buffer (pH 7.1) which was also 0.172 M in sodium chloride. The second digestion procedure was that used by Grabar (3) and also by us (7) for the digestion of horse antipneumococcus euglobulin. Ten cc. of a 1.4 per cent solu*ion of pseudoglobulin was dialyzed for 2 days in Visking tubing against cold acetate or citrate buffer, pH 4.5 to 2.0. The buffer solutions were also 0.2 M in sodium chloride. All hydrogen-ion concentrations were measured with a commercial glass-electrode sssembly. Twelve mg. of pepsin, crystallized by the method of Philpot (9), was then added to the dialysis bag, and the dialysis continued for 5 days at 4°C. The dialyzate was then changed to cold phosphateborate buffer (0.172 M in sodium chloride; pH 7.1). After 2 days’ further dialysis the solution was removed from the bag and ita volume noted. The protein concentration was determined by micro-Kjeldahl nitrogen analysis. The euglobulin digestions were carried out in the same way, except that 10 cc. of a 2 per cent solution and 16 mg. of pepsin were used. When the pepsin was added to the dialysis bag containing pseudoglobulin a t pH 4.15 a slight precipitate formed. At pH values of 3.96, 3.72, and 3.56 there was a heavy precipitate, as noted by Pope (10). A precipitate formed but disappeared on mixing a t pH values of 2.86 and 2.01. When the digests were dialyzed against the buffer at pH 7.1, these precipitates redissolved. Heavy precipitates formed in the euglobulin digeata at pH values of 4.5 to 2.7. At pH 2.1 the tendency to precipitate was less marked. Again these precipitates redissolved during the process of dialysis against buffer solution at pH 7.1. C . Sedimentation velocity measurements The original globulins and the digests were analyzed in the standard Svedberg oil-turbine ultracentrifuge at 60,OOO R.P.M. for their molecular maas spectra. Observetiom of the positions of the boundariea were made by the Lamm scale method and the observed scale-line displacements were plotted a g h t die-
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tances in the cell. Further details of this procedure have been described in a recent monograph by Svedberg and Pedersen (11) and in a more compact fashion by Carter (2). One or more curves from each experiment were analyzed according to generally accepted procedures. In our diagrams solid lines represent the observed scale deviations and broken lines indicate the resolution into separate components. Since there is so much overlapping of the maxima in the l i e displacement-distance curves, no great accuracy is claimed for this resolution. The areas under the resolved curves were measured with a planimeter. These areas are proportional to the concentrations of the several components. III. RESULTS
Two samples of beef pseudoglobulin were digested a t room temperature and a t pH 4.2. One contained a nearly homogeneous component with s20 = 5.5s and another with s20 = 3.4. The presence of a third component which had not broken away from the meniscus after 120 min. in the ultracentrifuge at 60,000 R.P.M. was indicated. The second sample showed the presence of a component with = about 4.9, with much inhomogeneous material both lighter and heavier in weight. One sample of beef euglobulin was digested a t room temperature. Under these conditions the digest wm very inhomogeneous in sedimentation and no characteristic velocity constants could be measured. Digestion a t 4°C. for 5 days at various hydrogen-ion concentrations gave much more homogeneous fragments for both pseudoglobulin and euglobulin. Some of the scale-line displacement-distance diagrams obtained with the digested pseudoglobulin after sedimentation for 125 min. a t 60,000 R.P.M. are shown in figure 1. The several components, which have sedimentation constants in the neighborhood of 7,5, and 3, have been designated aa s7,sb, and sa for convenience. Here s7 represents the normal pseudoglobulin and s6 and sa the products of cleavage. As the pH of the digestion solution is lowered, the s7 component decreases in amount and the s6 component increases. At pH values of 4.0,3.8, and 3.6,the diagrams are quite similar. At pH 2.6, however, (not shown in the figure) there is no s7 and very little ss remaining. The main constituent now is s8, and a very small amount of a still lighter component makes its appearance. At pH 2.0 there waa so little protein left in the dialysis bag that the solution waa too dilute to be analyzed in the ultracentrifuge. From the volume and protein concentration at the end of dialysis the total protein remaining in the bag waa calculated. Since 148 mg. of pseudoglobulin and 12 mg. of pepsin had been added to each bag, the per cent loss of protein by digestion to fragments small enough to dialyze out of the bag waa easily determined. The assumption waa then made that all the protein left in the bag waa represented in the scale-line displacement-distance diagrams, and waa equivalent to the total area under the curves. The proportion of each component present waa then calculated. This assumption probably introduces no
* Throughout this report sedimentation conatsnta are given in centimeters per second per unit of &Id X 10".
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very great error, since the curves start a t the bwe line. This indicates that there is no new protein boundary coming into the diagram. To be sure, at
FIQ.1. Beef serum pseudoglobulin before and after treatment with pepsin TABLE 1 Peptic digestion of beef serum globulins: per cent dkatribution PH
OLOBULINU
h t
-__ Pseudoglobulin. . . , , . . , , . , , .
Euglobulin. . . . . . , , , , . . ,
, ,
.
,
.
4.50 4.15 3.96 3.72 3.56 2.86 2.01
16 42
4.50 4.15 3.96 2.70 2.01
22 25 38 72 85
40 48 56 76 87
PER cmm DIBTFZBURON
-
-g1b.0
81
14 19 17 13 15 17
13 30 36 32 25 15
0
11
1
4
0 0 0 0 0 0
pH 2.86 a lighter component was found, but it amounted to only 3 per cent of the total. Table 1 and figure 2 show the proportion of each component present after digestion at the variouR hydrogen-ion concentrations. No correction
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ANALYSIS OF PEPSIN-TREATJOD SERUM GLOBULINS
could be made for the sector shape of the cell, because it was desirable to use the full depth of the cell to obtain maximum resolution of the sedimenting components. Similar diagrams for the euglobulin digestion experiments are shown in figure 3. The normal euglobulin contains a homogeneous component with sto = 7.1, and in addition much inhomogeneous heavier material. The exact amount of the heavier components could not be determined, since a t 60,000 R.P.M. they sediment to the bottom of the cell in a very short time. After digestion at pH 4.5 the sedimentation constant of the s7component is unchanged (see table 2), PER CENT
PH
4.5
Digested
Digested LOST
6-10‘5.4
s2.=,? su=3.3
Digested
UNCHAUOED
0
at pH 2.7
UNDETERMINED
Df~tonrr in
Cell
FIG.2 FIG.1 FIG.2. Peptic digestion of beef pseudoglobulin FIG.3. Beef serum euglobulin before and after treatment with pepsin
but a small amount of s5 has appeared. There is also present some s3 constituent which may be mostly pepsin. The pH 4.2 digest shows a decreased amount of the 57 component, and an increase in s5 and s3components. At pH 4.0 digestion proceeds still farther and here the lighter components predominate. Also, the curve no longer starts at the base line, because the solution now contains fragments so light that they have not left the meniscus after 125 min. of sedimentation. At pH 2.7 very little s7 is left; and at pH 2.0 (not shown in the diagram) there is no s7 and very little s6 component. The main constituent now has a sedimentation constant of 2.2. In the case of the euglobulin digestion experiments, interpretation of the molecular mass spectrum diagrams is not so simple as with the pseudoglobulin
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experiments. There is still a large amount of heavy protein, si*ao, present after digestion; this decreases with decreasing pH so that it probably contributes to the amounts of the various lighter components shown in the line displacement-distance diagrams. It waa not generally possible, therefore, to determine the percentage distribution of the non-dialyzable material which was left. At pH 2.01 the scale-line displacement-distance diagram after 15 min. of sedimentation was such that the small amount of heavy material remaining could be determined (table 1). It will be recalled that a t this pH the intermediate component s7 has completely disappeared. TABLE 2 Sedimentation constants of normal and digested .mum globulins PBOTPN
'EB cmm PBOTPN
-
-
x
1011
6s
3.4
6.5 4.9.
Beef pseudoglobulin: Normal. . . . . . . . . . . . . 1.48 Digested . . . . . . . . . . . . 0.M Digested pH 4.2, heated.. ...................... 0.87 Digested cold, pH 4.5.. .......... 1.22 Digested cold, pH 3.72.. ........................ 0.88 Digested cold, pH 3.72.. ....................... 0.44 Digested cold, pH 2.86. . . . . . . . . . . . . . . . . . . . . . . . . 0.37
D
3
E7
7.3
7.3 5.3 5.4 3.3 -
Beef euglobulin: Normal. ........................................ Digested cold, pH 4 . 5 . . ........................ Digested cold, pH 3.56. . . . Digested cold, pH 3.56.. ....................... Digested cold, pH 2.01
0.80 1.81 1.50 0.75 0.35
Horse pseudoglobulin (diphtheria antitoxin) : Digested pH 4.2, heated.. ...................... Digested cold, pH 4.08.. . . . . . . . . . . . . . . . . . . . . . . .
0.57 1.05
5.7 5.6
2.50 0.24
5.0 5.4
7.1 7.0 4.7 5.1 2.2'
-
Horse euglobulin (antipneumococcus antibody) : Digested cold, pH 4 . 5 . . ........................ Digested cold, pH 4 . 5 . . . . . . . . . . . . . . . . . . . . . . . . . .
* Approximate
values on inhomogeneous material.
The sedimentation constants for all the components found in these experiments are summarized in table 2. The values given for the undigested globulins are undoubtedly too low, since concentrated solutions were used. For the fragments sedimentation experiments were also made with dilute solutions, and values near the true constants obtained. Also included in this table are sedimentation constants obtained with some digested horse globulins. Horse diphtheria antitoxin, a pseudoglobulin, is split by pepsin in the cold a t pH 4.08 to give a substance with sS0 = 5.6. This value is in good agreement with the value of sgo = 5.7 which waa previously obtained with thk material after diges-
ANALYSIS OF PEPSIN-TREATED SERUM GLOBULINS
189
tion at room temperature and heating (6). Horse antipneumococcus antibody, a euglobulin, is split by pepsin a t pH 4.5 into fragments with 820 = 5.4 (8). IV. DISCUSSION
From figure 2 it is apparent that for this sample of beef pseudoglobulin the greatest amount of the ss component is obtained by digestion at pH 4. Digestion at higher acidity leads to the disappearance of more of' the 5' component, but the s6 component continues to be split into sa and then to dialyzable fragments. The constancy in amount of the sa component down to pH 3.56 is interesting. According to Philpot (9), pepsin has a sedimentation constant of about 3. It will therefore contribute to the area under the sa peak in the displacement-diitance diagram. However, the pepsin added amounts to only 7 per cent of the total protein, so that the remaining 6 to 12 per cent must be pseudoglobulin. In the digest a t pH 2.86 there ie also present a small amount of material lighter than the s* component. In table 1 this constant has been designated as sz, although its sedimentation constant could not be determined. Although molecular weights cannot be assigned to the several fragments on the basis of sedimentation velocity observations alone, and diffusion studies with such mixtures are impracticable, some approximations may be made. Normal serum globulins have a molecular weight of about 160,000. This is the component that we have designated as s'. The components with sedimentation velocity constank between 5.1 and 5.4 which are found could be accounted for if this basic globulin molecule were cut in half crosswise to give a product of molecular weight in the neighborhood of 80,000. The homogeneity of this component also suggests a cleavage into halves. In a study of the product formed when horse diphtheria antitoxin is digested at pH 4.2 a t room temperature, and then heated at 58"C., the physical constants, antibody activity, and carbohydrate content of the product all suggested that the active antibpdy molecule remaining after digestion was 60 per cent of the original (6). Digestion of horse antitoxin at pH 4.08 in the cold gives a product with the same sedimentation constant as that obtained previously (cf. table 2). On the other hand, digested horse antipneumococcus euglobulin with = 5.4 combines with twice as much specific pneumococcus polysaccharide as before digestion, as shown by Grabar (3) and others. It is homogeneous in sedimentation and seems to consist of halves of the basic unit which corresponds in physical behavior to our s7 component (8). The component s3 which has been found in the digested pseudoglobulin is probably one-fourth of the original molecule. With sedimentation constant szo = 3.3 it could have a molecular weight in the neighborhood of 40,000. The ss component of the euglobulin is present in rather larger amounts (cf. figure 3) and sediments a t a slower rate than does the pepsin. The sedimentation constant obtained for the digest a t pH 2.0 was 2.2. These fragments appear to be smaller than quarters of the original molecule. In one experiment, that with pseudoglobulin digested at pH 2.86, a smaller component appears after the sa boundary. This may possibly be a component intermediate between a quarter
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M A R Y L. PETERMANN
of the large basic unit and the fragments of molecular weight less than 10,oOO which would have been lost by dialysis. Slight but consistent differences were found between the sedimentation constants of the pseudoglobulin and euglobulin components. Whether these indicate differences in size, shape, or solvation was not determined. In previous studies of protein digestion made with the ultracentrifuge, two types of enzyme action have been found. Annetts ( l ) , working with papain and egg albumin, found two fractions in her digests: a light dialyzable fraction, consisting of polypeptides and amino acids, and a heavy fraction. This heavy fraction contained no unchanged egg albumin and its sedimentation constant was reduced from 3.5 to 3.2. Its electrophoretic mobility was greatly reduced. Tiselius and Eriksson-Quensel (12) digested egg albumin with pepsin in acetic acid. They also obtained a light, dialyzable fraction, but their heavy fraction appeared to be unchanged egg albumin. Increasing the time of digestion only changed the relative proportions of the two components. They suggest two ways in which proteins may be split: “(1) all molecules are simultaneously but gradually broken down to products which are no longer attacked by the enzyme, (2) only a few molecules are attacked in each time interval, but these are quite rapidly broken down to the end products. In the former case a digestion mixture should contain a number of products which would show a more or less continuous variation in size and other properties between those of the original and the end-products. In the latter case the mixture should contain unchanged large molecules and fully digested end-products, but no appreciable amount of intermediate substances.” The digestions described in this paper conform to the second type, in that, as with peptic digestion of egg albumin, the large molecules are apparently unchanged, a t least in sedimentation behavior. Digestion of both pseudoglobulin and euglobulin a t pH 4.5 causes no change in the sedimentation rate of the s7 component. However, in our digestions intermediate components are found, which show, not a continuous variation, but definite size classes. The best explanation of these results would be that the breakdown process which is rapid in acetic acid proceeds more slowly at higher pH values, so that the steps in the process can be followed. It is as if the degradation could be observed in a kind of slow-motion picture. The first step, at least, is so gentle that antibody function, which depends upon structure, is not disturbed. The action of proteolytic enzymes other than pepsin under conditions where digestion is incomplete have been studied mainly on antibodies. Pope (10) has obtained the same splitting of horse diphtheria antitoxin with trypsin, papain, and fibrinolysin. Xorthrop (4)has split diphtheria antitoxin with trypsin a t pH 3.7, and Parfentiev (5) has used a number of enzymes to digest the same material. It therefore seems probable that this manner of breakdown is a characteristic of the serum globulins and that fragments of the same sizes would be obtained in digestions with other proteolytic enzymes. Although in the light of Annetts’ work (1) the action of papain on egg albumin differs from that of pepsin, no significant differences are found when beef serum pseudoglobulin is treated with these two enzymes. The pseudoglobulin is broken down to give
ANALYSIS OF PEPSIN-TREATED SERUM QLOBULINS
191
new and homogeneous components with sedimentation constants of 5.3 and of 3.5 to 4.0 when it is digested with papain-cyanide at pH 5.0 and at 4OoC. These components are thus comparable in size to the ones obtained by pepsin digestion. These experiments will be described in detail in another place. From these studies it appears that, at hydrogen-ion concentrations removed from the optimum for proteolysis, pepsin splits beef and horse serum globulins into approximately halves and quarters, then into dialyzable fragments. The nature of the components does not change when the digestion is carried out a t successively lower hydrogen-ion concentrations, but the ratio of the components is shifted to favor the presence of lighter molecules. V. SUMMARY
1. Beef pseudoglobulin and euglobulin have been digested by pepsin a t hydrogen-ion concentrations well removed from the optimum for proteolysis. 2. After digestion at pH 4.2 at room temperature in 5 per cent sodium chloride solution much of the protein remains in solution on heating to 58'C. This soluble protein consists of several ill-defined components, with sedimentation constants of 5.5 and 3.4. 3. When digested at 4OC. a t pH 4.5 to 2.0 both pseudoglobulin and euglobulin form amounts of dialyzable material which increase with increasing hydrogenion concentration. 4. The non-dialyzable fractions remaining after digestion consist of unchanged protein and of new components with sedimentation constants of 5.1 to 5.4 and 2.2 to 3.3. 5. As the hydrogen-ion concentration approaches the optimum for peptic digestion the nature of these components remains the same, but the proportion of lighter molecules increases at the expense of the heavier components.
The author wishes to express sincere thanks to J. W. Williams for advice and encouragement in this work. REFEREKCES (1) ANNETTS,M.:Biochem. J. 90, 1807 (1936). (2) CARTER, R. 0.: J. A m . Chem. SOC.63, 1960 (1941). P.: Compt. rend. 207, 807 (1938). .(3) GRABAR, J. H.:Science 93, 92 (1941). (4)NORTHROP, (5)PABFENTIEV, I. A.: U.9. patents 2,065,196 (1936)and 2,123,198 (1938). M.L.,AND PAPPENBEIMER, A. M., JR.:J. Phys. Chem. 46, 1 (1941). (6) PETERMANN, (7) PETERMANN, M.L., AND PAPPENHEIMER, A . M., JR.:Science 93, 548 (1941). (8) PETERMANN, M.L.,AND PAPPENHEIMER, A. M., JR.: In manuscript. (9) PHILPOT, J. ST.L.:Biochem. J. 29, 2458 (1935). (10)POPE,C. G . : Brit. J. Exptl. Path. 20, 132 (1939). (11) SVEDBERG, T.,AND PEDERSEN, K . 0.: The Ultracentrifuge. Oxford University Press, London (1940). (12) TISELIUS, A , , AND ERIKSSON-QUENSEL, I.-B.: Biochem. J. 33, 1752 (1939).
