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can be explained on the assumption that the difference between the mean sedimentation rates is somewhat more than the sum of the standard deviations of the two distributions. Earlier studies indicating the advisability of correcting sedimentation constants of influenza virus preparations for solution viscosity, because of the presence in such virus preparations of variable amounts of a viscous impurity, have been confirmed, REFERENCES (1) FRANCIS, T . , JR.: Harvey Lectures 37, 69 (1941). (2) KNIGHT,C. A.: J. Exptl. Med. 80, 83 (1944). (3) LAUFFER, M. A., AND STANLEY, W. M . : J. Exptl. Med. 80,535 (1944). (4) SHARP,D. G.,TAYLOR, A. R., MCLEAN,I. W., JR.: BEARD,D., AND BEARD,J. w . : Science 100, 151 (1944). (5) SHARP,D. G.,TAYLOR, A. R., MCLEAN,I. W . , JR.,.BEARD, D., AND BEABD,J. w.: J. B i d . Chem. 166, 585 (1944). (6) STANLEY, W. M.: J. Exptl. Med. 78,267 (1944). (7) STANLEY, W. M.: J. Exptl. Med. 81, 193 (1945).
ON A LOW-DENSITY LIPOPROTEIN APPEARING I N NORMAL HUMAN PLASMA’ KAI 0 . PEDERSEN Institute of Physical Chemistry, University of Upsala, L’psala, Sweden Received August 8 , 1946
At the beginning of the 1930’svon Mutaenbecher ( 6 ) made a study of several normal sera in the ultracentrifuge. Amongst other things he found that when the same serum was investigated at different concentrations the ratio albumin/ globulin, calculated from the sedimentation diagrams, increased with increasing serum concentration. In 1933-34 McFarlane (5) studied a number of different dilutions of various sera, and obtained results in concordance with those of von Mutzenbecher. The same was also the case for “artificial sera” made from mixtures of serum albumin and serum globulin. No reasonable explanation for this concentration effect has so far been given. McFarlane also noticed the presence of a component with a sedimentation constant between thoee for albumin and globulin ;he named this the “X-protein.” It was generally most distinct in the concentrated human sera, but disappeared into the “albumin” peak in the dilute ones. Although the concentration of the X-protein in normal human serum amounted to 20-30 per cent of the total protein, McFarlane did not succeed in isolating this substance, nor did he observe 1 Presented at the Twentieth National Colloid Symposium, which was held at Madison, Wisconain, May 28-29, 1946.
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LIPOPROTEIX I N HVMAh’ PLASMA
it in the albumin or the globulin fractions obtained by precipitation with ammonium sulfate. A reexamination of McFarlane’s sedimentation diagrams from the normal globulin fractions shows, however, the presence of a slower moving component which McFarlane assumed to be serum albumin. According t o t h e author’s experience, serum albumin is not precipitated by half-saturated ammonium sulfate unless the pH of the mixture is adjusted t o 5 or below. A protein with properties in the ultracentrifuge similar t o those of McFarlane’s X-protein is precipitated, however, by 45-00 per cent saturated ammonium sulfate. Since McFarlrtne did not n-ash his precipitate, a small amount of albumin might have keen present in this in the form of mother liquor, but this cannot account for the large quantity of substance sedimenting more slowly than the normal glolulin, in one case 43 per cent and in another 22. The major part of this component must undoubtedly have consisted of the “X-protein.” Likewise, in the sedimentation experiments with the albumin fraction, some Xprotein surely must have been hidden in the “albumin peak.” I n the following, some studies on the nature of this X-protein will be given. THE JNFLUENCE OF VARIOUS SALTS ON THE APPEARANCE OF THE
X-PROTEIN
Several years ago, it was found by the author (4, 10) that the sedimentation diagram for human serum was extremely sensitive t o small variations in the salt concentration of the solution, especially when the concentration is close t o that prevailing under physiological conditions. A more systematic study with various salts was then started (8). Figure 1 shows the effect of varying the concentration of added potassium chloride. I t is seen that the diagram is most sensitive to small changes in the salt concentration when this is between 0.15 and 0.35 M potassium chloride. When the influence of the nature of the salt was studied further, it was found that some salts already showed very pronounced effects a t low concentrations, while others were apparently not capable, within reasonable salt concentrations, of affecting human serum. Simultaneous investigations on other sera-cow, horse, rabbit, and pig-did not reveal any salt effect. Different electrophoretically isolated fractions from human serum were also 7)investigated with respect t o the salt effect. It was found that the ( p globulin fraction contained one component with s20 ,- 7 S, another component whose sedimentation constant varied with the salt concentration (3 < s * ~< 5 ) , and finally some inhomogeneous material. When the 7-glotulin fraction was examined alone in the ultracentrifuge, it showed mainly the normal globulin sedimentation with s20 -7 S independent of the salt concentration. It can be concluded, therefore, that the labile X-protein is a &globulin (see 10, page 400). Attempts t o correlate the effect with chemical and physical properties of the salt solutions r e r e in vain until finally it was found that the whole effect was dependent upon the density of the solution. A given density would produce almost the same effect on the sedimentation diagram irrespective of the salts present. This means that the whole effect is due to a difference in specific volume between the albumin and the X-protein. It means also that when
+
158
KAI 0. PE3ERSEN
the sedimentation constant is calculated in the routine way, an incorrect value for s20 is obtained. I n figure 2 the routine calculated sedimentation constants for both the albumin and the X-protein @-globulin) peak have been plotted against the density of the solvent. For both peaks the line of regression has been determined. It is seen that the albumin line is almost parallel to the abscissa, as it should be if the right value for 1.' is used. (In fact, the correct value for albumin is V = 0.73&.) For the X-protein, however, s20 varies very strongly with the density, which means that its specific volume must be quite different from 0.75, and close
5
10
I5
5
IO
I5
5
IO
5
I5
60
IO
I5
O m m
30 -
PO
Ni
IO 5
IO
I5
LO
90-
PO
FIG.1. The effect of varying the concentration of potassiuln chloride on the spdimentation diagram for normal human serum, .411 diagrams recalculated to the same theoretical scale distance; 1-mm. exposures taken 130 to 115 min. after reaching full speed; mean temperatwe = 27°C.;Rerum concentration = 0.4 CO.
t o 1. As the line of regression for the X-protein crosses the abscissa between 1.03 and 1.04, it means that in solution with densities above 1.04 the X-protein will rise t o the top of the cell instead of sediment toward the bottom. The low density of the X-protein has been used in the isolation of this protein. THE IKFLUESCE OF THE SERVM COSCESTRlTIOS O X THE APPEARASCE OF THE
X-PROTEIN It has been found that the size of the X-protein peak, for a given salt concentration, depended upon the serum concentration. In order to study this phenomenon, a normal human serum and the corresponding plasma were dialyzed against a citrate-phosphate buffer of such a density that the X-protein and the
LIPOPROTEIS Ilr’ HUMAS PLASMA
159
albumin peaks could he easily measured separately from the sedimentation diagrams. A number of solutions \vith Concentration varying from about 2 per crnt t o about 5 per cent rvere then studied in the ultracentrifuge. Some of t h e given in figure 3,
FIG.2. The variation of S ~ for O albumin and &globulin (X-protein) with the density of the solution. I n this calculation a specific volume of 0.75 has been assumed for both proteins. 0 refers t o values obtained i n runs with serum. Serum concentration = 0.4 CO. 0 refers to values from runs on X-protein isolated as &scribed on page 160.
From figure 3 it is seen that the X-protein is not visible in the lowest concentration, but is very distinct in the 5 per cent solution. Prom figure 4 it is seen that the total protein concentration calculated from the sedimentation diagram increases proportionally with An, as it should do. For the albumin and the globulic, however, this is not the case; their increase is less than what could be expected. The X-protein. on the ot)her hand, does not start until An is about 0.0035, but its concentration then increases linearly ~ i t hAn of the solution. I t therefore seems likely that some albumin and globulin in the mor:: concentrated solutions enter into the X-protein complex.
160
XAI 0. PEDERSES
This view is also strengthened by the finding that if purified human globulin is added t o a fresh solution of human serum, the X-protein peak increases, whereas the normal globulin peak augments much less than expected. If the
FIG.3. Variation of sedimentation diagram of human serum with protein concentration’ The diagrams have all been reduced t o the same protein concentration and scale distance? so that they are strictly comparable. t gives the time in minutw after the centrifuge has reached full speed, b is the scale distance, and A n is the measured difference batween the refractive index of the protein solution and that of the buffer solution. X = X-protein; .4 = albumin.
Pb-3 Swum
~
o A16umn
xGlhlh
D
X-pmkm oTohl
A Albumn
Globulh
D
X-phm o Tohl
FIG.4 . Variation of the concentration of the individual components of human serum and plasma with An for the solution, as calculated from the sedimentation diagram. For the calculation of the protein concentration from the sedimentation diagram, it has been assumed that An = 0.00189 was equal to a protein concentration of 1 g protein per 100 ml. of solution.
serum is old or if a globulin other than human is used, the normal globulin peak is enlarged exactly as one would expect. ISOLATIOE: O F THE X-PROTEIK FROM HUMAN SERUM
As mentioned earlier, the low density of the X-protein may be used for its isolation. If 35 ml. of normal human serum is mixed with 25 ml. of saturated magnesium sulfate no precipitation takes place usually, but if this solution is spun in an air-driven centrifuge a t 27,000 R.P.M. for 6 hr., an oily layer will
LIPOPROTEIS IS BUMAX PL.4SM.4
161
collect at the top of the centrifuge tubes. This oil consists mainly of the Xprotein. It may be further purified by resuspension in half-saturated magnesium sulfate and respinning in the centrifuge, but a large amount of X-protein is lost by this procedure, probably owing to the splitting off of albumin and globulin from the X-protein. If such a purified X-protein solution is dialyzed against 0 2 Jf sodium chloride and stcdied in the cltracentrifuge, it shows a sedimentation diagram like the left-hand one in figure 5 ; the sedimentation constant for the main peak corresponds almcst t o that for albumin. If, however, the density of the solution is increased, as in the right-hand diagram, the peak splits into two peaks, one sedimenting as altumin and the other, marked 8, at a slower rate. A number of experiments have been carried out in solutions of X-protein isolated in the manner just described. I n figure 2 the routine values obtained for
20
IO
0
5
10
15
0
5
10
I5
FIQ. 5 . Sedimentation diagrams from runs with an X-protein isolated from human eerum. s20 for this p-globulin have been plotted against the density of the solution. The dotted line gives the line of regression for this component. From this a value of T * = 0.97 is calculated for the hydrated particle. The partial specific volume of the unhydrated particle is probably lower. From the line of regression n e obtain a value of $20 = 5.9 S in water. If it be assumed that the particle is spherical, we have a particle weight of about 1.9
x
106.
-A diffusion experiment, made on the same material, gave 0 2 0 = 1.7 X lo-’. From this value, sz0 = 5.9 S and J’ZO = 0.97, a particle weight of 2 . 6 X IO6 is obtained for the hydrated X-protein. The phosphoivs content of the X-protein is very high, and it has been estimated that between 20 and 45 per cent of the X-protein consists of phosphatides (8). These values are much higher than those given by Blix et al. (3) for electrophoretically prepared P-globulin. Recenbly, however, still higher values for the phosphatide content of a pl-globulin from human plasma have been found a t the Harvard Medical School (7), and this PI-globulin shows the same properties as the X-protein. In connection with the method of isolating the X-protein just described, it may be of interest to note the following: It has always been found (8) that in the range 0.5 to 0 . 6 saturation with ammonium sulfate (pH above 5) the precipitates m e very difficult to clear with the centrifuge. They generally rise to the top of the centrifuge tubes, and it is necessary t o filter the
162
KAI 0. PEDERSEN
solutions in order to collect these fractions. Electrophoretic examination showed that they were rich in PI- and pn-globulins, but in addition both albumin and a-globulin were always present. A similar low-density protein has recently been described by Adair and Adair (l),but this protein must, according t o their serological tests, probably have bcen a comparatively pure p-globulin. DISCUSSIOX
From what has been said above it appears most likely that the X-protein in human serum is a reversible dissociable compound consisting of p-globulin and variable amounts of albumin, y-globulin, and lipids. I n concentrated serum it will contain rather liirge amounts of albumin and globulin, but as the serum is diluted or the X-protein is purified, the albumin and the globulin are split off. If the albumin and t,he globulin are taken u p by the X-protein, one would expect that the sedimentation constant f ~rthe X-yo:ein compound would increase parallel with the incrcnse in its content of th? more dcnse proteins. So far, however, no such incrczse has ever bcen o’iser ;t..l. According to some recent experimen’s by Hlix and the author, the electrophoresis diagram for h1;man seruni is hardly c!ianged when the lipids are extracted according t o 131is (Z),but thc sedimentat,ion diagram is much altered in so far cs the X-prot,ein has completely dissppeared frbm the diagram. As a t the same time the alimmin peak has increased, it miist be supposed that, the main part of the p-globulin, after extraction, scdimenik at the ssme rate as the albumin. Thus in this case it is only the centrifuge that can tell us whether or not some change in the protein solution has occurred. The presence of the X-protein can only be demonstrated in fresh human serum. As the serum becomes old i t loses its ability t o form an X-protein. This sensitivity of the X-protein is after all due t o the lipid pwt of the compound, where reesterification and hydrolysi; may tdic place, thus giving rise t o new substances with different properties (e.g., lecithin + Ivsolecithin, etc.). Oxidation of the unsaturated fat)ty acid component of the phosphatides also seems possible. That changes, for instance in the lecithin part, actually destroy the Xprotein, both in serum and in the isolated state, has recently been shown by Petermann (9). She found that after treatment with lecithinase the X-protein component disappeared from the sedimentation diagram both of isolated Xrum. I n the first case both the albumin and the globulin peaks are twice as large after the treatment as before. I n the whole serum only the globulin peak was increased after the treatment. The amount of X-protein seems t o vary very much from individual to individual, hut for a given individual it seems t o be rather constant. There seems perhaps to be rather little X-protein in the blood from persons in the teens. So far no scra other than human have shown the presence of an X-protein. A s , however, it is known-for example, from the work of McFarlane (5)that the increase in the globulin pcak is less than proportional t o the increme in serum concentration, while the opposite holds for the “albumin” peak, it must be supposed that part of the globulin and the albumin, in these sera too, combine
LIPOPROTEIN IN HUMAX PLASMA
163
with the 8-globulin to form some kind of an X-protein; but its sedimentation properties are such that this component will be hidden in the “albumin” peak. In an investigation still in progress the author has found that although the fractions precipitated between 0.45 and 0.6 saturated ammonium sulfate may sediment at a rate equal t o that for serum albumin or slower, they show diffusion constants considerably lower than that for the albumin. Values as low as Dna = 3 X lO-’ have been observed. From the ease with which the X-protein takes up certain proteins and lipids it may be supposed that it plays a very important rble in the transport function of the blood. SUMMARY
The labile X-protein in human plasma, first described by McFarlane, has been shown to be a lipoprotein, 8-globulin, with density close to 1. The particle weight for this compound is of the order lo6. In concentrated serum the X-protein complex furthermore contains considerab!e amounts of both albumin and 7-globulin.
The expenses connected with this study have been defrayed by grants from the Iiobel Fund and the Rockefeller Foundation. ILEFERESCES (1) ADAIR,G. S., A K U A D A I R ,~ I L - R I E Y .L: J . Physiol. 102, l i p (1944).
(2) BLIX,G . : J. Biol. Chem. 197, 495 (1941). (3) BLIX,G . , TISELIL-S, A , , AND SVEKSSON, H: J . Biol. Chem. 197, 485 (1941). (4) JERSILD, h f . , A N D PEDERSES, K . 0 . : Acta Path. Microbiol. Scand. 16, 426 (1938). (5) SICFARLANE, A. S.: Biochem. J. 29, 407, 660 (1935). (6) ~ I C T Z E N R E C HP. ER YO , N : Biochem. Z. 266, 226 (1933). (7) ONCLEY, J . I,., SCATCHARD, G., AND BROWN, A , : J. PLys Colloid Chem. 61, 184 (1947). (8) PEDERSEN, K. 0 .: llltracentrifugal Studies o n Serum arid Serum Fractions. Almqvist and Wiksells, Upsala (1945). (9) PETERMAXN, MARYL . : J. Biol. Chem. 162, 37 (194G: (IO) ~ Y E D B E R Q T., , ASD PEDERSEN, E;.0.: The Cllraccidrijuge, p . 398. Oxford University Press, Xew York (1940).
