INDUSTRIAL
and
ENGINEERING
CHEMISTRY ANALYTICAL EDITION
+
Harrison E. Howe, Editor
The Ultracentrifuge and Its Field of Research THE SVEDBERG, University of Upsala, Sweden
pendent of the rubber plants, etc. Not only substitutes but entirely new products, such as cellulose derivatives, artificial resins, and other synthetic polymerization products are finding extensive use in the service of modern man. I n all these cases ire are dealing with high-molecular substances and colloids. It d?es not takedeep thinkingtoconclude that many indispensable articles of our daily life would be unsuitable and too expensive, if we did not know the right way to produce them, and this, in its turn, requires knowledge of high-molecular compounds. Three hundred years ago the first Swedish settlers tried to find their living on the shores of the Delaware, using very simple utensils and obtaining the necessary products from the cultivation of plants and animals. I n our days the same place is the site of powerful industries producing in a better and cheaper way many of the necessities of daily life.
C
OLLOIDS and high-molecular substances are the building stones of the cells and the various tissues of the organisms. Life is not possible without these substances, because no other material lends itself to the manifold performances here required. S o other material offers such a wealth of varieties, such pliable forms. Accordingly the biological and medical sciences which aim a t a n elucidation of the processes taking place in living beings are interested in the properties of high-molecular substances. Only by a detailed study of the behavior of these compounds will it be possible to find proper remedies in the case of disturbances. It is obvious, therefore, that our bodily welfare is highly dependent on our knowledge of the properties of the macromolecules of highmolecular compounds. Illness and death cannot be fought successfully if we do not know the chemistry and physics of the high-molecular material of our own body. When human beings began to improve their conditions by making weapons, various domestic appliances, and clothing they had to borrow from animals and plants to supplement what t h e i r o w n b o d i e s l a c k e d . Clubs of wood were used as s u b s t i t u t e s for the heavy paw of the lion and the bear, the hide of cow or sheep was swept around the body to protect the thin human skin. Thus was laid the foundation for the creation of the superbeing which modern man together with all his t e c h n i c a l facilities represents. The human facult i e s w e r e extended until nowadays man is a brain in the center of a superbody. The craftsman of o l d times had to use what his fellow creatures, the animals and plants, made. I n our d a y s powerful industries are busy producing artificial substitutes for many of t h e n a t u r a 1 materials. Paper makes papyrus and vellum superfluous, artificial silk makes it unnecessary to cultivate the silkworm, synthetic rubber makes us inde-
Development of the Ultracentrifuge The realization of the important role played by highmolecular compounds both in the life of the organisms and in many industrial processes has awakened a lively interest in systems of this kind and a number of new methods are being applied in their study. One of the new tools is the ultracentrifuge. Before d e s c r i b i n g t h e present forms a few historical notes may be permitted. The ultracentrif u g e o r i g i n a t e d in some work on colloids done in Upsala about 1920 concerni n g p a r t i c l e size in gold sols (68). We had tried to determine d i s t r i b u t i o n
FIGURE 1. ROTORS AXD CELLS 1-ppei. For centrifugal fields u p t o 710,000 times the force of gravity. Largest diameter, 10.4 cm.; mean active radius, 3 . 2 5 cm.; height of column of 'solution, 0.8 cm. Lower. For centrifugal fields up to 300,000 times the force of gravity. Largest diameter, 18 cm.; mean active radius, 6.5 cm.; height of column of solution, 1.8 cm.
113
114 INDUSTRIAL AND ENGINEERING CHEMISTRY VOL. 10, NO. 3 curves by recording the settling of the particles under the influence of g r a v it y . Only very coarse-grained sols (down to about 100 mp) could, however, be studied in that way. It was natural for me to turn my attention to the possibility of increasing the force acting upon the particles by using a centrifugal field instead of the field of gravity. Earlier attempts in this direction had not been very successful, however, and when I spoke to my research students about this possibility they were not very enthusiastic about it. It was not until I came to the University of Wisconsin as a visiting professor in 1923 that I f o u n d i n t e r e s t i n t h i s problem. J. B. Kichols, now at the du Pont Experimental Station in Wilmington, w a s w i l l i n g to cohperate with me. We built a small machine alloTying FIGURE 2. DIbGRAMXATIC REPRESENTATION OF OIL-TURBINE cLTR.ICESTRIFUGE optical observations of sedimentation to be carried out during rotation (G7). Contrary to expectations, we found that the solution of this The field was not more than about 1000 times gravity and protein is monodisperse and defined by the environment. One sedimentation could be followed only for a short period, owis therefore justified in speaking about its particle mass as its ing to convection currents. We felt confident, however, that molecular weight. This finding stimulated interest and one the problem could be solved. of the medical foundations in Sweden (Therese och Johan After my return to Upsala, Dr. Rinde and myself undertook Anderssons Rfinne) granted me a sum of money sufficient for a systematic study of the conditions of convection-free sedibuilding a high-speed ultracentrifuge to study the behavior mentation, using fine-grained gold sols as test objects ( 6 9 ) . of protein molecules in intense centrifugal fields. CollaboraI n the first place, we found that the sample of solution studied tion with Ljungstrom and Lysholm enabled me to obtain must be sector-shaped in order to permit the molecules to convection-free sedimentation in fields 100,000 times gravity travel along radii and not strike against the walls of the vessel in 1926 (66). I n the spring of 1931 further improvements of enclosing the sample. Secondly, the temperature of the the machinery accomplished by Boestad and me made poscolumn of liquid has to be kept constant both in space and sible sedimentation measurements at 200,000 times gravity time; otherwise convection currents caused by density differ(mean radius 5 = 65 mm.; height of column of solution = ences set in. We therefore spun our first rotors in hydrogen 12 mm.; 54,000 r. p. m., 51). Using the same radius and the at atmospheric pressure so as to reduce materially the heat same height of column of solution, we reached 260,000 times caused by friction against the surrounding gas. gravity early in 1932 (GI),300,000 in the spring of 1932 (56), I n 1924 we were able to perform faultless sedimentation and 400,000 in the spring of 1933 (49, 5 2 , 5 7 ) . in centrifugal fields 5000 times the force of gravity and to measEssentially higher fields cannot be utilized with rotors of ure the size distribution in gold sols down to the most finethis size because of failure of the material. It seemed of ingrained ones. The name ultracentrifuge was proposed for terest to try a smaller rotor type capable of giving considerthis new research tool. Using the same apparatus Fihraeus ably higher intensities although a t the sacrifice of height of and I (1925) succeeded in determining the particle weight of column of solution and homogeneity of the centrifugal field. hemoglobin by means of sedimentation measurements (64). Reducing the mean radius from 65 to 36 mm. and the height of s a m d e from 18 to 8 mni.. sedimentation measurements in fields' up to 600,000 times giavity were made in the fall of 1933 (55) and u p to 900,000 times gravity in the summer of 1934 (48,60). The rotors used in these experiments exploded, however, after a few runs. A further reduction of the mean radius to 32.5 mm. and improvements in the construction have made it possible to do regular measurements in fields u p to 710,000 times gravity (32). The comparison of measurements made in very intense centrifugal fields, using a low column of solution and a small mean radius, with measurements made in somewhat less intense fields using a higher sample situated farther from the center of rotation has shoxn that the accuracy is much better in the latter case, at least as far as sedimentation velocity measurements are concerned. Theoretical considerations (0.Quensel and K. 0. Pedersen) and experimental tests have shown that the pover of the ultracentrifuge to resolve a mixture of molecular species is DroDortional to w*& where w is the angular velocity, 5 the histance from the center of rotation, and h the height of FIGURE 3. SECTIOX OF CELLS column of solution (54). The largest value for this product Upper. Cell for optical observations reached so far is 5.83 X 10' (70,000 r. p. m., h = 1.65 cm., Lower. Cell for separation under optical control d
MARCH 1.5, 1938
ANALYTICAL EDITION
. OF OIL-TCRBIKE t FIGT-RE 4. AXIALSECTIOX
T
z = 6.58 cm.). For standard equipment, therefore, a large rotor is to be preferred. From the many different experimental machines built in Upsala, two standard types have been developed (48). The first is adapted for the region 500 to 15,000, and the other for the range 15,000 to 750,000 times gravity. The low-speed machine is driven directly b y a high-frequency motor and is provided with ball bearings. The rotation takes place in hydrogen a t atmospheric pressure and the casing is immersed i n a w a t e r t h e r m o s t a t . It is used for sedimentation equilibrium measurements in solutions of high-molecular substances and for sedimentation velocity measurements on heavy particles. O u r h i g h - s p e e d machine is driven by oil turbines and has white-metal bearings with movable, damped pistons. The rotor spins in hydrogen a t r e d u c e d pressure. It is used for velocity m e a s u r e m e n t s in solutions of high-molecular compounds and for equilibrium measurements on low-molecular substances. A few details concerning the oil-turbine ultracentrifuge may be of interest. The sample to be studied is enclosed in a sectorshaped cell provided with planeparallel quartz window (Figures 1 and 3 ) . Recently a cell type with a dividing membrane in the middle has been introduced for analytical determination of sedimentation.
The rotor (Figures 1 and 2) of chromium-nickel steel is supported by horizontal bearings, B , and B1, and kept in rotation by means of two small txvin-oil turbines, TIand 2'2, one on each end of the shaft. Hydrogen is admitted at the periphery and constantly pumped off so as to maintain a pressure of a b o u t 2 0 mm. Thermocouples Th, in the bearings and a radia-
115
tion thermocouple, Th,, near the rotor serve for temperature control of the centrifuge. -4 beam of light from a mercury lamp, L , filtered through Lf,,Lf2, and Lf3, passes the cell, C, in the rotor on its way to the camera. The exposures are timed by means of the electromagnetic shutters, SIand SI. For speed measurements and speed control a magnetic generator, M, is used in connection with a reed-frequency meter, an oscillograph, or a frequency bridge. The pressure oil which feeds the turbines is produced by a special oil compressor, cooled and thermostated to a suitable temperature before entering the turbine chambers. A system of channels in the heavj- steel casing makes it possible to thermostate the centrifuge by means of oil or water circulation. The lubrication oil for the bearings passes through an oil filter and is controlled by valve V,. By changing the speed of the motor which drives the compressor and by operating valve V 3 , the pressure of the oil entering the turbines may be regulated so as t o make possible sedimentation measurements at any desired speed beheen 5000 and 140,000 r. p. m. The resistance thermometers, etl, Rt?, and Rta, and the manometers, GI, G?,and GS, enable the operator to control temperature and pressure in various parts of the machinery.
~ ~ ~ A detailed section of the centrifuge proper through the axis of rotation is given in Figure 4.
Figure 5 gives a view of the installation showing bhe camera, the centrifuge on its foundation, the oil coolers, and the hand rails leading to the pit \There the motor, compressor, and filters are located. Recently a n air-turbine-driven, self-balancing ultracentrifuge has been developed by Beams ( 5 ) of the University of Virginia and improved and adapted to sedimentation measurements by Bauer (4)and by Wyckoff ( 7 ) a t the Rockefeller Institute in Sell- York. Here a light Duralumin rotor hangs
FIGURE 5 . OIL-TURBIKE CLTRACESTRIFUGE IKSTALLATION
~
INDUSTRIAL AND ENGINEERING CHEMISTRY
116
VOL. 10, NO. 3
FIGURE6. SEDIMEA-TATIOK PICTURES OBTAINEDBY LIGHTABSORPTIONMETHOD(LEFT) AKD CURZESOF COSCENTRATION DISTRIBUTIOAFOR LIMULUSHESCOCY.4NIA~ AT PK 6.8 (RIGHT) (ERIKSSOX-QUEKSEL) Le imentation constants of components, 56.5 X 10-13, 34.6 X 10-13, 10.1 X 70';3, and 5.9 X 10-13. Centrifugal force 120,000 times sravits. Time between exposures, 5 minutes ~
RIeniscus
Protein components
I 0 Index
I
I
I
I
I
I
1
0.2
0.4
0.6
0.8
t0
1.2
istance from Meniscus (x), cm. on a thin steel shaft arid ia suppoited by ail air-filni Ilearing. The friction, and consequently the energy consumption, are therefore very low. The air turbine is sealed off from the vacuum chamber in which the rotor moves by surrounding the vertical shaft with an oil-gland-shaped bearing. Beams has further succeeded in spinning electrically a hanging Duralumin rotor supported by an air-film bearing (6). These new types of ultracentrifuge promise to be of great service in many cases, although the resolving power has not yet been pushed t o the height obtainable with the steel rotor of the oil-turbine ultracentrifuge.
Sedimentation The process of sedimentation is in most cases followed by optical means. TWOdifferent properties of the solute may be utilized for the determination of the concentration distribution in the rotating solution-namely, the light absorption and the refraction. I n both cases the thickness of the layer of liquid studied necessitates long-focus lenses in order t o avoid parallactic errors. When using the absorption method, photographic exposures of the sedimenting column are made from time to time by light of a wave length absorbed by the solute. These pictures are then measured by means of a microphotometer and give the relation between concentration c and distance 2 from center of rotation. Each molecular species is brought out as a step on the c-3: curve (Figure 6). The change in refractive index can be used in various ways. The simplest way is to apply the Toepler schlieren method (80). The different molecular species present are then recorded on the plate like the lines of a mass spectrum (Figure 7). The most accurate procedure for obtaining the real concentration distribution in the sample studied is t o take pictures of a finely ruled scale through the sedimenting column of solution by light of a wave length which is not absorbed (18, 19). By measuring the displacement, z, of the lines, we get the concentration gradient, dc/dx, as a function of the distance from the center of rotation. Each molecular species is therefore shown as a maximum on the z-z curve (Figure 8). I n many cases, such as antibodies, enzymes, mixtures of proteins and carbohydrates, it would be of great value if a mechanical division of the sample studied could be accomplished after a certain time of centrifuging and controlled by optical observations. Analytical determination of sedimentation would then be possible. Experiments of this kind can now be performed using the cell with partition membrane shown in Figure 3 (81). Figure 9 demonstrates the completeness of the separation (pneumococcus antibody).
Ultracentrifuge Measurements
Two kinds of measurements can be made by means of the ultracentrifuge. In the first place, one may centrifuge long enough for a state of equilibrium t o be reached between sedimentation and diffusion. Then for each molecular (or particle) species the following formula is valid (53, 58) :
where M = molecular (or particle) weight, R = gas constant, 7' = absolute temperature, c = concentration of solute, V = partial specific volume of solute, p = density of solution, z = distance from center of rotation, and w = angular velocity. I n this way one obtains the molecular weight directly, independent of shape or hydration ( $ 2 ) . If several molecular species are present in the solution the molecular weight values calculated for different distances from the center of rotation show a marked drift. Freedom from drift is a criterion of homogeneity with regard to molecular weight. In the second place one may use a centrifugal field strong enough to cause the molecules or particles to sediment with measurable velocity. This procedure enables us to find how many different kinds of molecules are present in the solution. If the sedimentation velocity is referred t o unit field and water of 20" C. as solvent, it is called the sedimentation constant: dx/dt
s = - w2z 9/70
1
-
VPO
see. 1 - v-p
Meniscus
1
1 I
Protein components
Index
FIGURE 7. SEDIMENTATION PICTURES FOR LIMCLUS HEMOCYAKIS (ERIKGSOX-QUENSEL) Obtained by Toepler schlieren method a t p H 6 . 8 , showing the fastest three sedimenting components of s = 56.5 X 10-*3,34.6 X 10-13, and 16.1 X 10-18. Centrifugal farce 120,000 times gravity. Time between exposures. 5 minutes
AKALYTICAL EDITION
MARCH 15, 1938
0.15
i
O''O
n
N
0.0 5
65
GO
70
x, cm. FIGURE 8.
SEDIMENTATION
DIAGRAM FOR
HEMOCYANN (PEDERSEN)
LIhfOLUS
Obtained by Ilerefractive index method a t pH 6.8, showing the same four main components as i n Figure 6 a n d also a small amount of a fifth. Centrifugal force, 120,000 times gravity. Time after reaching full speed, 35 minutes
where 77 and p are the viscosity and density of the solution: qo and po the same quantities for water a t 20' C. By combining diffusion and sedimentation data the weight of the different molecular species is calculated according to the formula (53, 69)
where s = sedimentation constant, and D = diffusion constant. This equation may be deduced in such a way that the independence of M of shape and hydration becomes evident. Both in Equation 3 and in Equation 1 the molar frictional constant is eliminated, in the first case because two independent measurements are carried out, one on sedimentation and one on diffusion, and in the second case because sedimentation and diffusion are responsible for the state of equilibrium reached. Sedimentation measurements in the ultracentrifuge can also be used for the determination of the weight distribution or size distribution of molecules or particles in a polydisperse mixture (3, 21, 29, $1, 45, 69) As the theory is rather complicated, me will not go into it here.
FIGCRE 9. AXALYTICAL DETERMIXATIOX OF SEDIMZKTATIOX
Horse antibody serum against pneuMEASUREMENT OF DIFmococcus Type I polysaccharide. Left. Content of upper cell comI n order to calcupartment after addition of polylate molecular weight from saccharide. Right. Content of lower cell compartment after addition sedimentation velocity deof polysaccharide terminations, it is necessary to have a n independent and accurate measurement of the diffusion constant, D. I n many cases only a small amount of substance is available and a micromethod has therefore been worked out (18, 80).
FUSION.
The light from a lamp, b, passes filters, c, and a transparent scale, d, on its way to the diffusion vessel, f, and the camera, n. A thermostat ensures constant temperature. A diffusion cell with plane-parallel windows and requiring only about 1 cc. of solution is used (Figures 10 and 11). By means of a movable slide the solvent can be placed on top of the solution. The change of concentration with time a t the boundary between the two columns of liquid is then followed optically, by means of either the light absorption or the refraction method. ELECTROPHORESIS NEASUREVENTS. As a supplement to the study of the sedimentation of molecules in centrifugal
I
i'
204.5 c~--P
iw
I
I I
I I
Q
r
r
)
O
k e*
I
j g e ' d c
FIGURE 10. DIFFCSION APPARATUS (LAMM)
b
a
118
INDUSTRIAL AND ENGINEERING CHEMISTRY
fields it is of considerable value to be able to investigate their migration in electric fields (71, 76). The electrophoretic mobility is measured using the moving boundary method. I n a U-tube with plane-parallel n&~dows the solution to be studied is placed underneath the solvent and an electric potential gradient is created over reversible electrodes. The movement of the boundary is observed by means of the light absorption or one of the refraction methods. 9plot of the mobility as a function of p H furnishes two important data-viz., the isoelectric point and the mobility per p H unit in the isoelectric region.
VOL. 10, NO. 3
APPLICATIONS.The ultracentrifuge has a wide range of application. With the aid of this tool molecular weight determinations have been done from about 20,000,000 (tobacco mosaic virus) down to about 40 (lithium chloride). This technic offers the unique possibility of carrying out an analysis of the various molecular species or particle sizes present in a solution. The sedimentation constant is a very characteristic molecular property and, by means of it, it is often possible to follow sensitive aggregation and dissociation reactions in biological systems. The combination of sedimentation equilibrium and sedimentation velocity measurements allows certain conclusions with regard to the shape of the molecules or particles. This is often of importance when investigating high-molecular compounds. Among the substances studied so far are proteins, polysaccharides, polyhydrocarbons, polystyrenes, dyestuffs, and other synthetic organic compounds, as well as inorganic colloids and inorganic salts. Results of Protein Investigations
FIGURE 11. DIFFUSIOX CELL(LAMM)
Some of the main results of the protein investigations carried out in Upsala may be mentioned (48, 60, 62, 54). A very striking but rather unexpected property of protein solutions discovered by the ultracentrifugal analysis is the perfect molecular homogeneity. This means that the solution of a certain protein is either uniform with regard to molecular weight or contains a limited number of different molecular species, as a rule in equilibrium with each other. Change in protein concentration, in pH, or in concentration of other solutes present may bring about dissociation or association. If the sedimentation proceeds so quickly that no appreciable diffusion takes place during a run, the molecular homogeneity can be tested simply by studying the degree of sharpness of the receding boundary (Figures 13 and 14). I n cases where the sedimentation proceeds more slowly, so that noticeable diffusion occurs during a run, the homogeneity can be tested by comparing the theoretical sedimentationdiffusion curves with the observed ones (Figure 15). A homogeneity test may also be performed by means of sedimentation equilibrium measurements (Figure 16). Here the molecular weight values should be independent of t h e distance from center of rotation. The dependence of a protein on pH is exemplified by the stability diagrams of Helix pomatia, Helix arbustorum, Helix nemoralis, and Helix hortensis (Figure 17, 11).
The general setup for electrophoresis measurements is similar to that used for diffusion determinations. The migration apparatus has recently been very much improved (77). The straight limbs of the tube are now made rectangular in section, thus offering a larger surface for conducting away the heat. The front walls are plane-parallel, so as to allow accurate optical observations to be carried out. The limbs are divided into two parts which on both ends are cemented to precision ground-glass plates. Corresponding plates are also cemented to adjacent top and bottom parts of the U-tube. This makes i t possible to divide the column of solution after a suitable migration time. I n order to minimize the danger of thermal convection currents and a t the same time allow higher voltages to be applied, the electrophoresis is conducted at about 4" C. where water has its density maximum and where the change of density with temperature, therefore, is zero. A further feature of considerable importance f o r t h e analysis of mixtures consists in giving the w h o l e c o l u m n of l i q u i d in which the electrophoretic m i gration takes p l a c e a constant motion so as to prevent the boundaries from moving out from the straight limbs of the U-tube in long-timed experiments. To this end a n ebonite cylinder is slowly l i f t e d o u t of t h e l i q u i d in one of t h e electrode vessels b y m e a n s of c l o c k FIGERE 12. ELECTROPHORESIS CELL(TISELICS) work.
In the case of Helix p o m a t i a a n d Helix
nemoralis the protein contains only one com-
ponent at the isoelect r i c point, while the hemocyanin of Helix arbustorum and Helix hortensis contains two components in the isoe l e c t r i c region. On lowering or raising the pH, points are reached w h e r e a very small change in pH causes a great change i n t h e molecular state. The o r i g i n a l molecule of Helix pomatia of weight 6,740,000 (s = 98.9 X
10-13) dissociates stepwise into halves ( s = 62.0 X eighths (s = 16.0 X
and sixteenths (s = 12.1 x 10-13). The pH-dissociation products represent perfectly homogeneous com-
MARCH 15, 1938
ANALYTICAL EDITION
119
Meniscus
Protein
Index
FIGCRE13. SEDIMESTATIOS PICTCRES OBTAIKED BY ABSORPMETHOD(LEFT),AKD CURVESOF COSCEXTRATIOS DISTRIBUTIOS FOR HELIXHEXOCYASIS AT PH 5.5 (RIGHT)
TIOK
.M = 6,740,000: s = 98.9 X lo-'?. Centrifugal fnrce 45,000 times grarity. Time between exposures. 5 minutes. Sharpness of boundary a n d steepness of curves demonstrate t h e high degree a1 molecular homogeneity of this protein (Eriksson-Quensel!.
ponents. The presence of divalent ions (Ca--, M g - - ) causes a considerable change in the stability diagram of Heliz pomatin hemocyanin. hleasurements of the T p d a l l effect gave the first indication of this interesting phenomenon ( ' 1 ) . An analysis by means of the ultracentrifuge (Eriksson-Quensel) has sho1V-n that upon addition of 0.01 -If calcium chloride, the dissociation on the alkaline side of the isoelectric point does not become noticeable until a pH of about 9.5 is reached, where the molecule splits into halves and eighths. Kithout Ca - - the dissociation starts at pH 7.4.
FIGURE 14. SEDIMEST~TIOS OF HERIOGLOBIK ISCESTRIFUG~L FIELD900,000 TIMESGRAVITY (ERIKSSOS-QUESSEL) Time h e t a e e n expo-ure.
3 minutes
The reversibility of the dissociation-association process influenced by hydrogen-ion concentration is demonstrated by the following experiment (11) : A solution of Helix pomntia hemocyanin at pH 6.8 of sediimolecular weight 6,740,000) mentation constant 98.9 X was brought to pH 8.0, where it contains three components with the sedimentation constants 98.9 X 62.0 X and 16.0 X 10-13 (molecular weights 6,740,000, 3,370,000, and 842,000). The pH n-as then changed back t o 6.8 and a sedi-
Distance from Meniscus, cm. mentation analysis performed. It was found that all the fragments of dissociation had completely united to form the original component of s = 98.9 X (molecular weight 6,740,000). High dilution often causes dissociation. Thus, hemoglobin is partly dissociated into half niolecules upon dilution (5'6), In dilute solutions of thyroglobulin there are present several dissociation pioducts (23). The addition of an amino acid or another protein often causes dissociation (33). Thus serum alliuniin may be split by adding clupein (Figure 18). In certain cases even extremely small amounts of foreign substances map cause dissociation. Thus, the addition of 0.001 per cent thyroxin gives riie to an appreciable dissociation of thyroglobulin (24). The action of a dissociating compound on a protein is more or less specific. An amino acid xliich acts ptrongly upon a certain protein map have no effect on another protein, and vice versa. Thus arginin plus arninonium chloride dissociates serum albumin (Figure 19) but not Helix hemocyanin, while lysin plus ammonium chloride splits the latter protein but not the former (10,34). Guanidine chloride affects Helix hemocyanin very strongly but has only a very slight effect on seruin albumin. Clupein splits both, and arginin without ammonium chloride has no effect on either (10,33, 34). High salt concentration may cause dissociation or association. In solutions of thyroglobulin (s = 19.2 x 10-13, JI = 640,000), the addition of 4 M sodium chloride gives rise to a homogeneous association product of s = 196 X lO-'3, corresponding to a molecular weight of about 16,000,000 (23, 24).
Mer
PI
1
Distance from Meniscus, cm. PICTURES OBTAISEDBI ai^^^^^^^^^ NETHOD (LEFT)ASD CTRTESOF COSPESTRATIOK DISTRIBCTIOS FIGURE 15. SEDIUESTATIOS FOR u-L.kC'T.kLBL3IIS (Iof this protein.
Recently it has been found that a protein molecule may be split by the action of ultrasonic waves (8). Thus, Helix hemocyanin a t p H 6.2 is partly decomposed into half molecules. This process seems to be diiferent from the p H dissociation, in so far as a lowering of pH does not cause the half molecules to unite.
fL
Special Groups of Proteins
30
The above survey has ainied a t giving a general picture of the physico-chemical properties of the protein molecules, especially with regard to the influence of environment. I n the following, a short summary of some of the results obtained in Upsah for special groups of proteins will be presented. The serum proteins are among t h o s e which have been most fully studied, but which still p r e s e n t n o t a b l e difficulties Early sedimentation studies (27) in the ultracentrifuge shoved that in dilute normal seruw there are two main protein constituents with s = 4.3 and 7.1 corresponding to the albumin (S = 4.5, D = 6.2, N = 69,000) and globulin (s = 7.1j D = 4.05, AI1 = about 160,000) fiactions of the salting-out process a n d a s m a l l a m o u n t of a heavier globulin component of s = 18.5. In pathological sera new components often appear side by side with the noimal ones ( 2 7 )
-4 detailed study (BIcFarlane, Pedersen, and Tiselius) brought to light a number of new facts. It was found (26) that in concentrated sera, part of the globulin molecules dissociated and that this effect was probably due to the action of serum albumin (33, 34). The effect is different for different species. In Figure 20 sedimentation diagrams for normal human, cow, and horse serum are given. In diluted condition all three shorn the maxima of normal albumin and globulin, the globulin content decreasing in the order : horse, cow, man. I n the undiluted sera the globulin maximum is very much depressed and there appears in one of the diagrams (human serum) a new maximum (the “Xcomponent,” 25) probably corresponding to half or fourth molecules of globulin (t?$). In the horse and corn sera this dissociation product is hidden in the albumin maximum. A comparison of normal human serum with pathological sera reveals a number of intwesting differences (Figure 21, 2/71,
100
8C
60
20 1
I
5
I
I
I
7
9
II
2
/
4
I
I
1
6
8
10
~
!
Jfelzl: pomntza
I
80
40
c t
I
I
(j-&, I
0 0
1
I
I
I
I I
6
0
iielzs nemoralis
io
L
2
! )
4
I
1
6
1 8
Helix hortensis
FIGVRE17. PH-STABILITYDIAGRAIIS (ERIKSSON-QUEWEL)
I 10
J 12
MARCH 15, 1938
ANALYTICAL EDITIOS
In the first place the globulin content is usually very much increased, as is also the s-component. Sometimes new coinponents appear (Figure 21, malignant tumor of bile duct). I n the case of certain diseases of the kidney (Figure 21. nephritis) the albumin maximum is unsymmetrical, indicating the presence of niolecules of lower molecular Feight than qeruni albumin (possibly dissociation product.). The gloliulin
TlM€:l00min. a/ferjullspeed SCALE DISTANCE: 6 cm.
1
122
content often increases during a disease, probably as a result of increasing immunization (Figure 21, scarlatina). The possibility of using ultracentrifugal analysis for diagnostic purposes is being tested by McFarlane a t the Lister Institute, London. Ultracentrifugal studies of immune sera and purified antibodies ( I S ) have shown that the antibody activity is carried by a globulin component of one of the sedimentation conztants found in normal sera. Thus the antibody to Type I11 pneumococcus polysaccharide obtained from rabbit serum and containing 90 per cent specifically precipitable protein gave the normal serum globulin sedimentation conqtant
02
B h
N
0.1
, 6.00
650
7.01
x, cm. FIGURE 18.
6.0 6.5 7.0 Distance from Axis o f R o t a t i o n , cm.
8EDIJiEh-TATIOX DIAGRAM O F 8 E R C V h L B U h l I S
Is 2'6 PER CLrpEIN (PEDERSES) Rapidly sedimenting main maximum, A , represents undiasociated protein; slowly sedimenting maximum, B , is dissociation product s 1 x 10--3 and M - 1/8 t h a t of serum albumin. Sedimentation of clupein itself has been subtracted from curves.
-
i
h
X
IN
CM.
FIGURE
19. SEDIILIEhTATION DIAGRAM
O F SERUhl
ALBIZJYININ 2.6
PERCEST ARGISIXSOLUTION AT PH 5 (PEDERSES) Left. X i t h o u t ammonium chloride Right. After addition of ammonium chloride t o 0.1 M .
INDUSTRIAL AND ENGINEERING CHEMISTRY
122
VOL. 10, NO. 3
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IN
615 CM.
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CM I.
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FIGURE 21. K. H. B.
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6'52
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