4 64
SERGE N. TIMASHEFF AND ROBERT TOWNEND [CONTRIBUTION FROM THE EASTERN
REGIONAL RESEARCH LABORATORY,’ PHILADELPHIA,
Vol. 83 PENNSYLVANIA 1
Molecular Interactions in P-Lactoglobulin. V. The Association of the Genetic Species of P-Lactoglobulin below the Isoelectric Point2 BY SERGE N. TIMASHEFF AND ROBERT TOWNEND RECEIVED JUNE 23, 1960 The association reactions of the genetic species of b-lactoglobulins A and B have been studied between pH 3.7 and 5.2. Ultracentrifugal, electrophoretic and light scattering measurements have shown that 90% of @-Acan tetramerize in this PH range, the maximum of the reaction being between pH 4.40-4.65, while p-B cannot form aggregates greater than a dimer. I n mixtures of the two genetic species, mixed tetramers are formed. Previously reported equilibrium constants for this reaction were confirmed. It was shown b y direct fractionation t-hat 10% of @-Ais inert toward tetramerization.
Introduction
Material.-The genetically different ,+Ixtoglobulins used in this study were samples kindly given us by Dr. Aschaffenburg, as well as material prepared by us from the milk of individual cows. Before the preparation of @-A and p-B, the milk of ii number of cows of various breeds was screened by the paper electrophoresis technique of Aschaffenb~rg.’*~The screening study was carried out in collaboration with members of the Dairy Cattle Research Branch, U. S. Department of Agriculture and has been reported elsewliere. The p-A and 6-B were prepared from the milk of individual cows mainly by the method of Aschaffenburg.8 Sincc this method includes a step in which the protein is exposed to PH 2, at which it is known to dissociate into molecular subunits,IO this method was compared with that of Palmer1’ which involves no such step. For this cornparison milk from a single milking was divided into two portions and 8lactoglobulin was prepared from each portion by one of the techniques. T h e proteins obtained were found to crystallize with equal facility. Comparison of their ultracentrifu-
gal patterns a t PH 2.0 ( 2 5 O ) , 4.65 (2’) and 5.3 (25’) arid their electrophoretic patterns at p H 4.65, 5.3 and 5.6 revealed no difference between the two preparations either in p-A or p-B. This was taken as evidence t h a t protein prepared by the Aschaffenburg procedure is not altered. Methods.-The ultracentrifugal experiments were carried out in a Spinco Model El2analytical ultracentrifuge at 59,780 r.p.m., using Kel-F cells. The ultracentrifuge was equipped with a temperature control unit and a phase plate. Most electrophoretic experiments were performed in a PerkinElmer Model 38-AI2 apparatus at ice temperature. I n the study of the @-A subfractions, the electrophoretic experiments were performed on a Spinco Model H electrophoresis-diffusion apparatus’2 at l.Bo, using a n 11 ml. cell. Ares analyses were carried out planimetrically from enlarged projected tracings. Electrophoretic mobilities and sedimentation constants were measured with the help of a microcomparator. Light scattering measurements were performed in the Brice photometer13 equipped with 2 mm. slit optics; operations were carried out according t o the modified Dintzis technique,14as described previously.’ -411 pH’s were measured at 2 5 O , using a Beckmann Model G PH meter.12 Protein concentrations were determined spectrophotometrically at 278 m r using a value of 0.96 liter g.-’-cm.-’ for the absorptivity6 of both genetic species which are known to have identical absorptivity ~ a 1 u e s . l ~ Fractionation of p-A.-(3-Lactoglobulin A was dissolved in a 0.1 N NaC1 solution at 25’. This solution was adjusted t o pH 2.0 with N HC1 and kept two hours at t h a t PIT. The pH was then adjusted t o 5.10 with JV NaOH and the solution dialyzed against several changes of distilled water according to the usual 0-lactoglobulin recrystallization procedure. dfter two days, the heavy oil which came out of solutioti was separated by centrifugation and decantation of the supernatant. Both fractions were then seeded with :t crystal of 0-A. A heavy crystalline precipitate came rapidly out of the supernatant, while the oil slowly turned t o a crystalline mass. Approximately two-thirds of the protein was fouiid in the oil, the remainder in the supernatant fraction. Analysis of the Data.-All ultracentrifugal, electrophoretic and light scattering data were analyzed by methods described in papers 1 1 6 and 1 1 1 6 of this series. Theoretical light sc:rttering curves aud “component” distributions under ultracentrifugal and electrophoretic “reaction boundaries”16 were culculated using equilibrium constantsl7 previously
(1) Eastern Utilization and Development Division, Agricultural Research Service, United States Department of Agriculture. (2) This work was presented in part a t the 133rd National hleeting of the American Chemical Society, San Francisco, April 1958, and the 135th Meeting, Boston, April 1959. (3) R. Townend and S. N. Timasheff, Arch. Biuchern. Biophys., 63, 482 (1956). (4) S. N. Timasheff and R. Townend, THISJOURNAL, 80, 4433 (1958). ( 5 ) R.Townend, R. J. Winterbottom and S. N. Timasheff, i b i d . , 82, 3161 (1960). (6) R.Townend and S. N. Timashed, i b i d . , 83, 3168 (1960). (7) R. Aschaffenburg and J. Drewry, Nature, 176, 218 (1955). ( 8 ) R. Aschaffenburg and J. Drewry, Biochem. J . . 65, 273 (1957). (9) R. D.Plowman, R. Townend, C. A. Kiddy and S. N. Timasheff, J . Dairy Sci., 42, 922 (1959) (Abst.). (10) (a) R. Townend and S. N. Timasheff, THIS JOURNAL, 79, 3fi13 (1957). (b) R. Townend, L. Weinberger and S. N. Timashe5, i b i d . , 82, 3175 (1960). (11) A. H. Palmer, J . Biol. Cliem., 104,359 (1934).
(12) Mention of a specific commercial product does not constitute an endorsement by the United States Department of Agriculture. (13) B. A. Brice, hl. Halwer and R. Speiser, J . Opt. SOC.Amcr., 40, 768 (1950). (14) S. N. TimasheiI, H. M. Dintzis, J. G. Kirkwood and B. D. Coleman, THIS J O U R N A L , 79, 782 (1957). (15) C. Tanford and Y.Nozaki, J . B i d . Ckcm., 234, 2874 (1959). (16) L. G. Longsworth, in M. Bier, “Electrophoresis,” Academic Press, Inc., New York, N. Y.,1959, p. 91. (17) For the sake of clarity, the previously deduced values of t h r equilibrium constants6 for the reaction 48 # 84 are presented: 4.14 4.40 109 2.1 X 1011 6.9 X 10” 4.90 5.10 10” 2 . 2 x 1010 3.4 x 108 , T,OC.: 2.0 4.5 8.0 p H 4.65: 4 5 x 1010 2 0 x 10” K,: 5 . 5 x 10” T. OC. 15.0 25.0 pH 4.65: K.: 6.0 X 109 4.5 X los
In previous communications3+ it has been reported that /3-lactoglobulin undergoes a reversible aggregation a t close to 0’ in the p H region between 3.7 and 5.2] with a maximum a t p H 4.404.65. From an analysis of ultracentrifugal, electrophoretic and light scattering data obtained with preparations differing in their contents of the genetic species of this p r ~ t e i nit, ~was concluded that this association proceeds to the stage of tetramer.6 These results were consistent furthermore with the concept that 90% of 0-lactoglobulin A (@-A) can tetramerize, that P-lactoglobulin B (0-B) itself does not yield a tetramer and that in the presence of &A, approximately 30% of 0-B can enter into the tetramerization reaction forming mixed aggregates. It is the purpose of this paper to present a verification of these conclusions from experiments carried out with the separate genetic species of this protein.
Experimental
{
{
P-LACTOGLOBULIN : ASSOCIATION OF GENETICSPECIES
Jan. 20, 1961
deduced from light scattering measurements on p-A and 8-B mixtures of various compositions.eJ8 I t was considered t h a t only 90% of 8-A can tetramerize. The second virial coefficient at PH 4.65 was taken as zero.6
Results and Discussion Stoichiometry of the Aggregation.-Ultracentrifuga1 analyses of 0-A and 0-B were carried out a t 2” between pH 3.7 and 5.2 in 0.1 ionic strength acetate buffers. In this pH range, P-A gives a bimodal sedimentation distribution similar to that previously reported for the preparation containing both s p e c i e ~ , ~ Jwhile J ~ P-B gives only a single peak over the entire pH range even a t concentrations as high as 90 g./l. The PH dependence of the “component” distribution under the @-A “reaction boundary” is shown in Fig. 1. Like pooled 0-lactoglobulin, 0-A gives maximal aggregation a t pH 4.40-4.65; however, P-A shows the greater percentage of “heavy component” over the entire pH range concerned. The concentration dependence of the fraction of total area present under the rapidly sedimenting peak has been reported previously. I t was found to be consistent with the concept that only 90% of the protein can aggregate. In a similar way, electrophoretic experiments on @-Awere carried out as a function of protein concentration a t pH 4.65. The results, presented in Table I, show that, as the protein concentration increases, the amount of “fast component” increases. TABLE I ASSOCIATION OB &LACTOGLOBULIN A.” AREADISTRIBUTION IN ELECTROPHORESIS IN #H 4.65 ACETATEOF 0.1 IONIC STRESGTH.1’ Protein concn., d1.
-Rapid
5.2 10.0 16.0 16.3 20.4 ” 90% aggregable material.
Obsd.
71 79 86 85 87
component, %Calcd. ace. to Gilbert theory
74 82
85 85 86
The ultracentrifugal and electrophoretic data were analyzed in terms of the Gilbert theory of moving boundaries in an aggregating system.bv20 The calculated percentages of “rapid component” are shown as the filled circles of Fig. 1 for the f H dependence and by the last column of Table I for the concentration dependence. In both cases, good agreement is obtained between the predicted values and the experimental data. This is strong evidence for the correctness of the equilibrium constants previously reporteda and underlines the validity of the method of component analysis described in paper I.18 Although P-B has been shown not to give aggregates this large4 under conditions a t which P-A tetramerizes, i t was of interest to check the degree to which i t is capable of forming mixed aggregates with @-A. For this purpose, ultracentrifugal and electrophoretic analyses were carried out a t (18) S. N. Timasheff and R . Townend, THISJOURNAL, 89, 3157 (1960). (19) A. G. Ogston and J. M. A. Tilley, Biochcm. J . , 69, 644 (1955). (20) G. A. Gilbert, Discussions Faraday Soc., No. 2, 1955, p. 68; Proc. ROY.Soc. (London), AND, 377 (1959).
465
- oG ‘
f-”.
> a w
X
=
THEORY
40
I
a* I‘
466
SERGE
p
A
J3- 8
PROT
N.TIMASHEFP
CONC
AND
KOBERTTOWNEND I
3 ,
6 4 G/L
A
dL
8 7 13
60
-J--L.-
-.-.A-
70 30
53
.-f--L-
I----
50 5 0
6 6
L
n
A
AL
A
JL-
100
Xp
K)
-A
70
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9 2
d
4
h
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*o "I+ I 1
.O
II
I
20
IO
PROTEIN l
much lower than in the ultracentrifugal experiments. It can be seen however, that on the descending side in @-A and all the mixtures, the boundaries are bimodal. As the relative amount of 6-B increases, the fraction of the area under the rapidly migrating peak decreases. This indicates that the rapid peak is due to the formation of the tetramer, which is completely absent in 100% P-B. On the rising side, a hypersharp boundary appears in all cases. Quantitative analysis of these electrophoretic data is complicated by the fact that in this 9H region P-B undergoes a type of transition similar to that observed by Cann for other proteins.21 This gives rise to an anomalous appearance of the electrophoretic boundary and renders very difficult any analysis by such methods as the Gilbert theory. Qualitatively, however, the data of Fig. 2 are in good agreement with the concept that a fraction of P-B can enter into mixed association with &A. According to the Henry equation,22the mobility (u)is related to the charge Q (in e.s.u.) by
,-
2
0
Fig. 2.-Electrophoretic patterns of @A-B mixtures; r / 2 = 0.1 acetate buffer PH 4.65. Starting positions shown by vertical lines, 8000 sec. at 9.86 v /cm.
i
CONCENTRATION,
I
I
30 G IL
Fig. 3.-Analysis of light scattering data on &lactoglobulin A a t PH 4.65 in r / 2 = 0.1 acetate buffer. Experimental points: 0, 4.5'; 0 , 8 " ; A, 15'; A, 25"; lines are theoretical curves calculated as described in the text.
asgregating conditions (room temperature) the
Z M of &lactoglobulin a t pH 4.65 has been iound t o be +7.23 Using equation 2, this gives a ZT for
+
+
52, or 12 per monomer unit. the tetramer of The charge dzerence of +5 per monomer unit is too great, however, to be explained in terms of the assumptions on the molecular shape and charge distributions which are inherent in the Henry equation. It is possible that some specialized configuration of charges exists so that when monomer units combine, sites with local attraction for protons or sodium ions may be e s t a b l i ~ h e d . ~ ~ As a further check on the stoichiometry of the aggregation, light scattering measurements were carried out on p-A, P-B and a 1:1 mixture of the two as a function of temperature a t pH 4.65. The results obtained with @-Aare presented in Fig. 3. The symbols are experimental points determined a t 4.5, 8, 15 and 2.5". The lines are theoretical curves calculated assuming 90% of the protein aggregable. The excellent agreement obtained bears out again the earlier analysis of the data from mixtures of various compositions. The data a t 4.5" on @-A,P-B and a 1: 1 mixture are shown in Fig. 4. The dot-dash line is the best where r is the radius of the spherical molecule, curve drawn through the points obtained with q is the viscosity of the medium, and K is the Debye/3-B at 25". It is found to be a horizontal line with Hiickel- parameter. Q can be replaced by Ze the points (not shown on the Fig.) equally distribwhere Z is the mean net charge of the protein moleuted about it. Since a t 25" P-B does not aggregate cule and e the electronic charge. I n the case of to any extent, the data can be taken as proof that @-A, the electrophoretic mobilities of the peaks in under these conditions the second virial coefficient the descending limb corresponding to monomer is equal to zero, confirming reasoning used (mol. wt. 36,000) and tetramer (mol. wt. 144,000) previously6 in arriving a t an the assignment of zero and 4.1 X have been found to be 1.2 X cm.2 set.-' volt-', respectively, a t pH 4.65.6 t o the slope. The open circles are the points obIf monomer and tetramer are represented by equiva- tained with P-A a t 4.5" (also shown in Fig. 3), . parameters in eq. the triangles are points obtained with a 1: 1 mixlent spherss, r~ = 4'ja r ~ All 1 save r , 2 and u are identical for the two species, ture of P-A and 8-B. The dashed lines are curves and the ratio of the radii is a constant. Taking the calculated assuming that 90% of P--4 can tetraradius ai of the supporting electrolyte ions into merize, while 30y0 of P-B can also do so when mixed with /3--4. The quantitative agreement account, we get obtained in two cases establishes the correctness of the previously proposed stoichiometry of the reaction, ie., the aggregate is a tetramer (mol. wt. SubstituLion of valugs for the various parameters 144,000); 90% of ,B-a can aggregate; 30% of gives: ZT = 7.53 ZM. By titration under non(23) Y.Nozaki, L. G.Bunville aud C.Tanford, Tsrs JOURNAL, 81, (21) J.
R. Cam, THISJOURNAL,
80, 4263 (1958).
a-2) 0 , 6,Hoaty, P m . RW. SOIL (
t ~ n d ~ALiaa, ) , io6
(ioai).
6523 (1959). (24) C. Tanford, ibid., 10, 6840 (1867).
P-LACTOGLOBULIN : ASSOCIATION OF GENETICSPECIES
Jan. 20, 1961 4 . 5 0 ~ p~ 4.65 I
o
i
8-A
--......-
o,i r/2
ACETATE
I
I
MONOMER-TETRAMER PROGRESSIVE
ASSOCIATION
1
2 0
20
c
I
467
'
I
/I
TETRAMER,
Y
,
,
MONOMER
~
._.__._....... L.. I
20 TOTAL
I 0
IO
PROTEIN
I
I 20 CONCE,.TRATlJN.
30 G./L.
Fig. 4.-Analysis of light scattering data on p lactoglobulins a t p H 4.65, r/2 = 0.1 acetate buffer, 4.5". Experimental points: 0, @-A; A, 1:1 8-A-p-B mixture; 0 , p-B; dashed lines, theoretical curves for monomer-tetramer aggregation; dotted lines, theoretical curves for progressive association; dot-dash line best curve through experimental points on p-B a t 25'.
Fig. 5.-Species
PROTEIN
CONCENTRATION,
30 G /L
distribution for aggregation of p-lactoglobulin A, calculated by eq. 4.
be represented by K1 2P
82 Ki
__
+ P _J K2 83 + P Pa
82
P3
(4)
Kz = K1'14 the P-B molecules are capable of reacting with @-A, forming mixed tetramers. I n the case of Expressing concentrations as mole fractions, the P-A, as will be shown below, this effect is due to over-all equilibrium constantI6K , (for the 4 p d @ 4 molecular heterogeneity. With P-B no molecular reaction), is 3.43 x 1Ol6; then, if the four bonds are heterogeneity has so far been demonstrated. How- considered to be identical as a first approximation, ever, @-B appears to undergo a molecular trans- Kz = 9.26 X lo7, K1 = 1.92 X lo4. The filled formation in this 9 H range,25and it is possible circles in Fig. 4 are the light scattering points that prior to entering into the association reaction, obtained with 0-B a t 4.5" and are in fair agreement P-B must undergo an isomerization (P-BdP-B *, with the theoretical curve for dimerization of 3070 the equilibrium constant, K i s o m , of the unimolecu- of the protein, In the case of P-A, the direct monolar reaction being around 0.35-0.55). The over- mer-tetramer and progressive aggregation cases all reaction scheme is are indistinguishable. This is a result of the relation between the equilibrium constants in eq. 4. 4PA (PA)4 (10% 6-A not reactive) The calculation of species distribution for this case (Fig. 5) shows that dimer and trimer are never present in significant amount. The amount of trimer present is zero within the limits of ac(3) curacy of the present calculations. It is for this reason that tetramerization results in two ultra(with part of PB not reactive) (pB)z centrifugal peaks. If dimers and trimers were n P A + mpB r'(PA)n(PB)m; n m = 4; m I 2 present in appreciable concentrations a single peak would be obtained. 2o It is unreasonable to expect that the aggregation Light scattering and ultracentrifugal experireaction can proceed by the simultaneous collision ments have clearly demonstrated that the asof four molecules. Therefore, calculations have sociation of ,&lactoglobulin attains a limit when the been carried out for the case of progressive ag- tetramer stage is reached. If all the bonds are gregation via the dimer and trimer stages. Since, considered to be equivalent and the bonding is as will be shown below, there are good reasons to taken as occurring a t specific sites on the molebelieve that the tetramer is in the shape of a closed cule, @-Amust be a t least divalent with respect to ring, it has been assumed that it results from the the aggregation reaction. However, since no formation of four identical bonds; then, the addi- large three-dimensional networks are formed, it tion of the last monomer molecule would have a is extremely unlikely that &A could have more much greater free energy change than the forma- than two reactive sites per molecule. If the bindtion of either of the two bonds between the first ing sites on a P-A molecule were a t opposite ends three monomer units. This would result in a molec- of the molecule, large linear polymers of the proular distribution strongly favoring tetramer over tein might be possible. These, however, cannot other aggregated species. The dotted lines on be induced to form even at very high protein conFig. 4 have been calculated for this case which can c e n t r a t i o d ; the sites must then be located on the molecules so that the formation of aggregates (25) R. Townend and S. N. Timasheff, Abstracts, 185th Meeting, Am. Chem. SOC.,Boston, 1959, p. 18-C. greater than tetramers is sterically hindered. The
''
+
SERGE N. TIMASHEFF AND ROBERT TOWNEKD
468
4
Fig. B.-Schematic
Vol. 83
\ -
model of the tetramerization of 8-lactoglobulin A.
Green and Aschaffenburg model26 of the P-lactoglobulin molecule is composed of two identical spheres, with the 36,000 molecular weight species having an axial ratio of 2:l. It follows, then, that the reactive groups responsible for the aggregation must be symmetrically distributed on the double sphere molecule, and the cyclic tetramerization of P-A must result in a compact structure. If the bonds form, for example, a t 90” angles, the end result would be a closed ring tetramer, as shown in Fig. 6. In mixtures of the two species, P-B can participate in mixed tetramers, although by itself it can form no aggregate greater than dimers. Such a situation could possibly be due to the presence of two types of interaction sites on each molecule of P-B, one being capable of interaction only with P-A. This is not too unrealistic if the structural difference between &A and P-B should result in a steric effect hindering P-B bond formation a t the second site. Work on this phase of the problem is currently in progress. I n Fig. 7 is presented the dependence of the sedimentation constant on concentration for 0-A and P-B at 2 O , pH 4.65. Analysis of the data by the previously described method5 yields 6.80 S for the sedimentation constant of the tetramer a t infinite dilution. This is slightly higher than 6.40 S obtained from the pooled milk p r o t e k 6 I n that case, however, the data analysis was complicated by the presence of 34% of non-aggregable protein, Thus, the present value can be considered as more ~ - ~an ~ f/fo value accurate. 40% h y d r a t i ~ n ~gives of 1.33. This corresponds to an axial ratio of 1:3.7for an oblate ellipsoid of revolution and of 3.5: 1 for a prolate ellipsoid, in agreement with the concept of a compact structure. I n the case of P-B, calculation of the average . ~ a function of concentration was value of ~ 2 0 as carried out20.10assuming that 30% of the protein can dimerize. As can be seen in Fig. 7 this curve falls very close to that for non-aggregating protein,30 and it becomes impossible to detect ultracentrifugally such a degree of dimerization. The pH dependence of the aggregation suggests very strongly that carboxyl groups play an important role in the binding mechanism, since the pH maximum of aggregation corresponds to the pK of carboxyl ionization. This is further supported by experiments which show that such carboxyl-blocking agents as lead ions can strongly (26) D. W. Green and R . Aschaffenburg, J . Molecular Biology. 1, 54 (1959). (27) T.L. McMeekin and R . C. Warner, THISJOURNAL, 64, 2393
(1942).
(28) D. W. Green, A. C. T. North and R. Aschaffenburg, Biochcm. Biophys. Acta, 21, 583 (1956). (29) S. N. Timasheff and V. Luzzati, unpublished low angle X-ray scattering measurements. (30) J. P. Johnston and A. G. Ogston, ?‘runs. Faraday Soc., 4¶, 789 1946).
3
d rn
!
L -
0
-
I
1
20
40 PROTEIN
I
60
CONCENrR4T10tv.
1
80
G /L
Fig. 7.-Dependence on concentration of the sedimentation constants of 8-A and 8-B a t pH 4.65,2’. Experimental points: 0, 8-A; 0 , 8-B; solid lines, theoretical curves calculated with the Gilbert theory for tetramerization of 90% of 8-A and dimerization of 30y0 of 8-B; dot-dash line, concentration dependence of the @-lactoglobulinmonomer30; dotted line, theoretical curve for tetramerization of &A (equilibrium only); dashed line, deduced concentration dependence of the sedimentation constant of the tetramer (hydrodynamic effect only) (the upper solid line represents the sum of the two effects).
interfere with the aggregation. 3 1 The only presently known chemical difference between the two species is the presence in 0-A of two more carboxyls than in P-B.16 It would seem desirable, however, to refrain from further speculations until more detailed information is available on the nature of the 18,000 mol. wt. subunitsg of the two species and more definitive statements can be made concerning the structure of these molecules. Fractionation of P-A.-In all the data analyses, i t has been assumed that P-A contains 10% of a material which cannot aggregate. While this assumption seems to be in quantitative agreement with all the experimental data, it is based strictly on the observation by Tombs32that P-A can be resolved into two fractions by partial precipitation with salt. These differ in the amount of rapid “component” in electrophoresis a t PH 4.65. Tombs, however, presented no direct evidence for the effect of the two fractions on the aggregation, and it is well known that in this PH range a number of proteins give complex electrophoretic patterns.3 3 I n the course of studies on p-lactoglobulin A a t low pH’s (1.5-2.0),it was observed by us that on subsequent recrystallization, two fractions are obtained, one coming out as an oil, the other remaining in solution and generally separating out only on seeding. (No such effect is observed in P-B, which crystallizes out normally after exposure to pH 2.). This procedure was applied to the sample of P-A given us by Dr. Aschaffenburg and the resulting materials were analyzed ultracentrifugally and electrophoretically a t pH 4.65. The results of these measurements are presented in (31) R. Townend and S. N. Timasheff, unpublished experiments. (32) M. P. Tombs, Biochcm. J . , 67, 517 (1957). (33) See, for example, R. A. Brown and S. N. Timasheff in AMBier, . “Electrophoresis,” Academic Press, Inc., New York, N. Y.,1969, p. 817.
P-LACTOGLOBULIN : ASSOCIATION OF GENETICSPECIES
Jan. 20, 1961
TABLE111 PERCENTAGE OF RAPIDCOMPONENT IN &LACTOGLOBULIN A pH 4.65, r / 2 = 0.1 ACETATE BUFFER (ASCHAFFENBURG PREP.) Fraction
Electrophoresis (1') g./L Calcd. Obsd.
Original 16.3 85.0" .. Supernatant 12.3 Oily 1 2 . 8 93.gb Assuming 90% aggregable aggregable.
.
I
2
UNFRACTIONATED SUPERNATANT
4
OILY
I
FRACTION
469 0-A
1.50 1.45 I. 2 4
0.90
Sedimentation (2") Calcd. Obsd.
g./L
85.0 20.5 83.5" 67.0 21.6 .. 91.2 2 1 . 8 93.0b material. Assuming
.
*
84.5 62.5 89.2 100%
Table 111in which the percentage of the area found experimentally under the rapid component is compared with that calculated with the Gilbert theory.20 In both sedimentation and electrophoresis an assumption that 90% of the starting material is aggregable yields quantitative agreement between the theoretical and experimental values. In the case of the oily fraction, the amount of aggregable material has increased although not to lOO% as is shown in the last line of the table (cu. 96% seems to be a more realistic value). The supernatant has been enriched with the nonaggregable material. The Iack of complete purification in the oil is probably due to occlusion of non-aggregable material in it. A second treatment of the oil did not result in further purification, as now all the protein precipitated out on dialysis against distilled water. This also can serve to show that the non-aggregable component is not a product of the acid treatment. A second sample of P-A, prepared in our Laboratory by the Palmer procedure,l* was subjected to an identical treatment. The descending electrophoretic patterns a t pH 4.65 of the original p-A and the two fractions are shown in Fig. 8. The important point to note is the change in area under the slowly migrating portion of the boundary. This area is greatest in the supernatant and smallest in the oil. Since, according to Gilbert, this area should remain constant for a system in aggregating equilibrium, the excess area in the cases of the unfractionated and the supernatant materials must be due to the presence of non-aggregable material. The fact that the area under the 'slow "component" does not change with concentration for the oily fraction points to the absence of significant amounts of non-aggregable protein. These results establish beyond any doubt that P-A contains 510% of material which cannot aggregate and prove that the electrophoretically different components
u
Fig. 8.-Superposition of descending electrophoretic patterns of 8-lactoglobulin A fractions, r / 2 = 0.1 acetate buffer pH 4.65, 21,600 sec. a t 4.87 v./cm.; protein concentrations in g./100 ml. given in figure.
of Tombs32are related to the association reaction. This justifies, then, the last assumption made in the analysis of the aggregation data. Conclusions.-The data and its analysis presented above establish definitely that @-lactoglobulin A is composed of two fractions, one (90%) which can aggregate to a tetramer, the other (10%) being unable to aggregate. P-Lactoglobulin B can form a dimer with 30% of the protein reacting. This may be the result either of molecular heterogeneity or of an isomerization which precedes aggregation. In mixtures of the two proteins, mixed tetramers can be formed with not more than two molecules of 6-B participating. The equilibrium constants, previously deduced from studies on complex mixtures, describe quantitatively the aggregation of the individual genetic species of this protein. The tetramer appears to be in the shape of a closed ring with the bonds being formed a t specific sites symmetrically located on the molecules. It is highly probable that carboxyl groups are involved in the bond formation, but a detailed analysis of the nature of the bonds requires further information on the structure of the subunits of the two p-lactoglobulins. Work to that effect is presently in progress in our Laboratory. Acknowledgments.-We would like to thank Dr. R. Aschaffenburg for giving US samples of @-lactoglobulins A and B, as well as Drs. N. D. Bayley, R. D. Plowman and C. A. Kiddy for giving us milk samples from individual COWS used in the preparation of the genetically different P-lactoglobulins.
