Sonderabdruck aus der ZEITSCHRIFT FUR NATURFORSCHUNG Band 15b, Heft 12, 1960 Verlag der Zeitschrift fiir Naturforschung, Ttibingen Interaction and mixed aggregation of proteins from Tobacco mosaic virus strains By Saryaprata Sarkar Interaction and mixed aggregation of proteins from Tobacco mosaic virus strains By Satyaprara Sarkar Aus dem Max-Planck-Institut fiir Biologie, Abt. Metcuers, Tiibingen, CorrensstraBe 41 (Z. Naturforschg. 15 b, 778 —786 [1960] ; eingegangen am 10. Oktober 1960) Proteins from four strains of TMV, namely vulgare, flavum, dahlemense and Holmes’ rib grass, were electrophoretically examined singly and in pairs at different hydrogen ion concentrations. In weakly alkaline media an average of six polypeptide chains of TMV-protein remain asso- ciated together, while constant dissociation and reassociation takes place. This dynamic state of equilibrium is responsible for transient reciprocal associations of proteins or polypeptide chains from the first three TMV-strains. On lowering the pry value, these interacting proteins form mixed aggre- gates with intermediate mobilities. Protein from the fourth strain, Holmes’ rib grass, when tested against the strain, vulgare, neither showed any interaction in alkaline media nor formed mixed aggregates. Factors determining the formation of mixed aggregates and the possible relationship among the four strains have been discussed. The protein-part of a single Tobacco mosaic virus 157* amino acids (Wirrmann and Braunirzer, 1959)? particle consists of 2130440 polypeptide chains whose sequence has been recently worked out? which are very probably identical with one another! (Annerur et al., 1960). (Franky, Caspar and Kuve, 1959). A polypeptide * chain of the common TMV strain, vulgare, has a According to a recent letter from Professor W. M. Sraney addressed to Professor G. Mrtcuurs a polypeptide chain molecular weight of about 17500 and consists of of TMV protein contains 158 amino acids and not 157 as found here earlier. This result could now be confirmed also by Dr, H. G. Wrrrmaxy. 1 R.E. Frayxun, D.L.D. Caspar and A. Kiva, in: Plant 2-H. G. Wirrmann and G. Brausrrzer, Virology 9, 726 [1959]. Pathology, Problems and Progress 1908—1958, Univ. of 5 A, Anperer, H. Unuic, E. Weer and G, Scurama, Nature Wisconsin, Pp. 447—461 [1959]. [London] 186, 922 [1960]. INTERACTION AND MIXED AGGREGATION OF PROTEINS The polypeptide chains of several other TMV strains differ more or less in the composition and sequence of their amino acids ** but they all possess one remarkable common property: they can asso- ciate near their respective isoelectric points in an orderly way producing cylindrical protein-coats, typical of intact TMV-particles which were studied electrophoretically by Kramer and WITTMann, 1958 8, as well as Kuzczkowsx1, 1959 7). The capa- city is inherent in the structure of the polypeptide chains themselves since cylinders of variable length are produced even without the presence of a ribo- nucleic acid core. At neutral and moderately alkaline py the TMV protein has often been reported to exist as small aggregates of six polypeptide chains with a mole- cular weight of about 100000 and has been given the name “A-protein” as it was obtained by alkaline splitting of TMV. The size of an aggregate is, how- ever, a function of such factors as py, ionic strength, temperature, protein concentration and so on. On high dilution, at high alkalinity or by the action of various organic solvents, the protein falls apart into single polypeptide chains which undergo easy denaturation (AnpERER, 19598, Ansevin and Laur- FER, 1959 9, Wirrmann, 1959 1°). On the basis of these observations, it seemed interesting to investigate: 1. Whether in a mixture of proteins from two different TMV strains an exchange of polypeptide chains occurs, and 2. whether mixed aggregates can be formed on acidification of such a mixture of proteins. These investigations were undertaken with a view to finding out how far an exchange of polypeptide chains and formation of mixed aggregates are deter- mined by the number of charged groups per poly- peptide chain and by their structural similarities and differences. Four strains of TMV, vulgare, flavum, dahle- mense and Holmes’ rib grass, were available, of C. A. Kntent, J. biol. Chemistry 171, 297 [1947]. H. G. Wirrmany, Vergleichende Strukturuntersuchungen an Tabakmosaikvirus-Stimmen verschiedenen Verwandt- schaftsgrades. Communication to the meeting of the Deut- schen Botanischen Gesellschaft, Kéln 1960 and Virology 12, No. 4 [1960]. 6 ©. Kramer and H.G. Wirtmann, Z. Naturforschg. 13b, 30 [1958]. 7 A. Kieczxowsk!, Virology 7, 385 [1959]. 8 A. Anperer, Z. Naturforschg. 14b, 24 [1959]. 779 which the amino acid compositions are now known (Wirrmann, 19605). Flavum and vulgare resemble each other very closely; each polypeptide chain of the former contains one aspartic acid less and one alanine more than the latter and most probably one amide group more than the latter on the basis of electrophoretic measurements (Kramer and Wirr- mann, 19581). As compared to both of them, dah- lemense shows several quantitative differences and contains one methionine per polypeptide chain which is not present in the other two strains 5, Holmes’ rib grass strain differs very widely from all the three above both qualitatively and quantitatively. Each polypeptide chain of Holmes’ rib grass contains several glutamic acid residues, three residues of methionine and one histidine, the last named being absent from the other three strains (Knicut, 1949 4, Wirrmann, 19605). Exchange of polypeptide chains and the forma- tion of mixed aggregates were detected by the method of free electrophoresis since the proteins of the four strains have characteristically different mobilities (Kramer and Wirrmann, 1958 8), Present observations speak for mutual interaction and mixed aggregation between proteins from closely related strains of TMV. I. Material and Methods Isolation of Virus. After multiplication on green- house grown tobacco plants each strain of virus was isolated in a pure form by the conventional method of differential centrifugation with alternate freezing and thawing. A preliminary removal of associated plant pro- teins from expressed sap was achieved either by heat- denaturation at 60°C for 10 minutes or by an isoelec- tric precipitation at py 4.3 (Commoner et al., 195017; Wirtmann 1959 13). As the isoelectric point of Holmes’ rig grass strain lies near about 4.5 (Oster 1951 *4; Ginoza and Atkinson 1955 45), only a heat-denaturation treatment was adopted for the primary clearing of the virus containing plant-sap. Splitting of virus and purification of A-protein. About 10 cm? of a 1—3% virus suspension in dilute phosphate 9 A.T. Ansevin and M.A. Laurrer, Nature [London] 183, 1601 [1959]. H. G. Wirtmann, Experientia [Basel] 15, 174 [1959]. E. Kramer and H. G. Wirrmany, Communication No. 2-25, Proceedings of the 4th International Congress of Bioche- mistry, Vienna 1958. 12 B. Commoner, F.L. Mercer, Pu. Meru and A. J. Zorn, Arch. Biochem. Biophysics 27, 271 [1950}. 19H, G. Wirrmany, Z. Vererbungslehre 90, 463 [1959]. 14 G. Oster, J. biol. Chemistry 190, 55 [1951]. 18 W. Gunoza and D. E. Arxtnson, Virology 1, 253 [1955]. 780 buffer (m/50) of py 7 was dialysed with stirring against one litre of glycine-NaCl-NaOH buffer of pu 10.3 — 10.4 (pu 10.6 in case of HR-strain) and an ionic strength of 0.1 at +4°C for 12 to 16 hours. The unsplit or in- completely split TMV particles still present were cen- trifuged down in the cold in a Spinco preparative ultra- centrifuge at about 60000¢ for 60 minutes, and the clear supernatant was dialysed for 12 to 24hours against glycine-NaCl-NaOH buffer of py 9.4 and ionic strength 0.04. The protein was purified by the method of countercurrent electrophoresis from the nucleic acid fraction at py 9.4 using the 10cm? standard cell of a “Phywe” electrophoresis apparatus. A potential gra- dient of 9—10 Volt/em for 8 to 10 hours was employed. As a criterion of purity, the ratio of absorption at 260 and 280 mu was kept as low as possible. The values for the proteins of the four strains abbreviated as AV, AF, AD and AHR were 0.54; 0.56; 0.58 and 0.53 respec- tively indicating that the protein-preparations were prac- tically free of nucleic acid (ef. Kramer and Wrrrmann 1958 ®), Concentration of protein was calculated from the ex- tinction at 280 my, since a good agreement exists be- tween Aggy and nitrogen content. A sedimentation ‘co- efficient of Syy FW 4 at py 8 was measured for each type of A-protein. Electrophoresis studies of A-protein mixtures at dif. ferent py-values, Electrophoretic mobility measurements were done at 3.2°C bath temperature in a “Phywe” electrophoresis apparatus. Errors in the magnification factor of the Schlieren camera and in the cross- sectorial area of Tiselius cell have been eliminated by using the sate limb of the same cell for all com- parative runs (AvBerty and Marvin 1950 1). To avoid smal] fluctuations in py or ionic strength a large volume of buffer solution was prepared for a whole series of experiments, kept at 4°C and checked for py and con- ductivity from time to time. AV, AF and AD solutions, each containing 0.4% pro- tein and AHR containing 0.25% protein were dialysed separately in Michaelis buffer of py 8.0 and of an ionic strength, /’=0.037. This ionic strength, which is half of that used by Kramer and Wittmann (1958) & was chosen to achieve greater numerical differences among ithe mobility values of the A-proteins and con- sequent better separation of the Schlieren peaks. The combinations and concentrations of the protein mixtures are given in Table 1. Each mixture was studied electrophoretically within one hour from the moment of mixing of the respective A-protein pair. After the runs the solution was recovered from the Tiselius cell and after standing for a week at 4°C reexamined electrophoretically. For aggregation experiments, the first three mixtures of table 1 were dialysed successively against Mich- aelis buffer of py 6.5, 5.5 and 5.0 (7 =0.037) for four hours each and then overnight against acetate buffer of py 4.92, = 0.037, all in a cold room at +4 °C. The 8 R.A. Arserty and H.H. Maarvin, J. phys. Colloid Chem. 54, 47 [1950]. S. SARKAR Concentration ': . of each protein Mixture type Pu in the final mixture {gm/100 ml] 1 AV4+AF 5 800 | 0.2 2 AVI AD 8.0 0.2 3 AF4S AD 8.0 0.2 4a AV + 4HR 8.0 0.13 4b AV+ AHR 8.5 0.12 Table 1. Protein mixtures used for electrophoretic studies. mixture 4a was acidified slowly by dialysis against buf- fers of gradually lower py values and examined electro- phoretically at every stage. As AHR precipitated out easily at py 5.0 and also for reasons described later, the mixture 4b was dialysed directly against a Mich- ae lis buffer of py 5.2, /'= 0.037. The aggregated protein mixtures were split again into the constituent A-proteins by dialysis against glycine- NaCl-NaOH buffer of py 10.4 and their electrophoretic diagrams at py 8.0 and 8.5 were compared with their corresponding original mixtures. II. Results The ascending Schlieren pattern was sharper than the descending one but the deviation from enantiography was not too great when each protein was present alone. Anodic mobility values, expres- sed as (4/sec)/(V/cm) are given in table 2. Each value is the average of several determinations with a potential gradient of 7.5 to 9.5 V/cm. The values were remarkably uniform with fluctuations mostly within + 2%. The descending peak has generally a_ slightly lower mobility value, a phenomenon of common experience (ALBERTY, 1953 17), The ascending boundary of AHR at py 8.0 has, however, a lower mobility than the descending one. An explanation for this discrepancy has to be sought in the nature of the AHR molecule itself, since all other factors are kept constant. However, the higher mobility-value of the descending boun- dary of AHR at py 8.0 might be due to dissociation of the imidazole ring of histidine. This amino acid is not present in the other three virus strains. It is also worth noting that the difference between the mobilities in the two arms disappeared by simply raising the py-value to 8.5. 17 R. A. Auperty, in: The Proteins, vol. I A, edited by Neu- rath and Bailey, Academic Press 1953, P. 523. INTERACTION AND MIXED AGGREGATION OF PROTEINS Mobilities of the observed peaks with the mix- tures 1, 2, 3 and 4b (cf. table 1) are given in table 3 and the electrophoretic patterns are re- produced in fig. 1. In all mixtures, excepting AV + AHR, the mobilities of the observed Schlie- ren peaks are different from those of the pure com- ponents as presented in Table 2. Fig. 1. Ascending-limb electrophoretic patterns of TMV-pro- tein mixtures. V, F, D and HR stand for proteins from vul- gare, flavum, dahlemense and Holmes’ rib grass strains re- spectively. The first three diagrams are at py 8.0 while the V+#HR mixture is at py 8.5. Protein type Pu U ascending | U descending AV : 8.0 0.46 0.46 AV 8.5 0.47 0.47 AF 8.0 0.37 0.36 AD 8.0 0.59 0.57 AHR, 8.0 0,67 0.71 AHR | 8.5 0.84 0.84 Table 2. Anodic mobilities (U) of A-proteins in Michaelis buffer, [=0.037, U=(u/sec)/(V/cm). 781 The Schlieren peaks were always much better resolved at the ascending boundary. Due to incom- plete separation and rapid spreading at the de- scending boundary. only one average mobility- value of this boundary has been given in some cases. From Table 3 it can be seen that: (1) mobi- lity of a particular peak remains practically un- changed even after 7 days’ standing of a protein mixture, (2) aggregation and resplitting does not affect the subsequent Schlieren pattern at pq 8.0 and 8.5, (3) a fall in total protein concentration from 0.4% to about 0.1% after aggregation and re- splitting hardly affects the mobility values and the Schlieren patterns. A comparison with table 2 indicates that (4) in all the three mixtures of AV’, AF and AD the mobility of the observed peaks are not exactly the same as the corresponding proteins when each is present alone; the two peaks appear to approach each other so to say, while (5) the mobilities of AV and AHR are exactly reproduced in mixture. Different degrees of flattening of the depicted. the sharpest AV + AHR and the most overlapping and spread- ing is AF + AD. On reducing the py value of the mixed solutions to 4.92 as described earlier, the proteins were found diagrams are being to aggregate as was evident from the opalescence of the samples. In table 4 are given the anodic mobilities (U = (4¢/sec)/(V/em)) of aggregated pro- teins both alone and in mixture at py 4.92. Agere- gated dahlemense protein moved as a single boun- dary while vulgare as well as flavum each formed two distinct peaks. According to Kramer und Wirr- mANN (19358) ® the faster peak, designated here as the main gradient. corresponds to rod-like aggre- | i - Total pro- i ; ; : Mixture | tein cone. | PH Time after | U ascending U descending ype gm/100 ml] mains P AF + At 0.40 8.0 1 hour 0.40 + 0.44 0.37 + 0.41 AF+ A} 0.40 . 8.0 7 days 0.40 + 0.45 0.38 + 0.42 AF + AV 0.11 | 8.0 : 12 days * 0.39 + 0.44 0.43 AV+ AD 0.40 : 8.0 1 hour 0.51 + 0.56 0.53 AV + AD | 0.40 \ 8.0 | 7 days 0.52 + 0.55 0.50 + 0.53 AV+AD 0.19 8.0 10 days * 0.52 + 0.56 0.50 + 0.53 AF+ AD | 0.40 8.0 1 hour 0.44 to 0.50 + 0.587 0.33:0.46 + 0.49+ AF+AD 0.40 30 | 8 days | 0.41 to 0.49 + 0.56+ 0.34 + 0.47 AF + AD 0.18 8.0 i 10 days * 0.41 to 0.48 + 0.57+ 0.35:0.46 + 0.51 AV + AHR 0.25 8.5 | 1 hour 0.47 + 0.85 0.45 + 0.83 AV+ AHR 0.25 | 8.5 : 7 days | 0.47 + 0.84 0.46 + 0.84 Table 3. Anodic mobilities of peaks observed in A-protein mixtures (4/sec)/(V/cm). * After aggregation and resplitting. * The slower peak is flattened and broader than the faster peak. 782 gates of dimensions comparable with intact TMV- particles, while the slower (= second) gradient cor- responds to discs and small incompletely aggregated units. The failure of better reproducibility as seen in table 4 may well be traced back to the high py-mobi- lity dependence at this py range (KRAMER and Wirrmann, 1958 §). , ae | U = (ulsec)/(V/em) at py 4.92 No. | Composition ascending j “descending | main second | main second peak peak j; Peak | peak 1 AF 0.77 0.68 | 0.64 | 0.65 2 AP 0.73 0.66 | 069 | — 3 AV 0.99 0.91 0.99 ; 0.97 4 AV 0.95 0.84 0.92 — 5 AD 1.09 _ 108 2 — 6 AD 1.09 ~ 110 | — 7 AF — AV 0.91 | 0.82 0.94 | 0.88 8 AV+AD] 097 | — 0.96 | — 9 AF+ AD ; O91 2 — 0.89 | — Table 4. Anodic mobilities of aggregates of A-protein and their mixtures. Results indicate that in all the three cases mixed aggregates are produced. The formation of one single gradient from AF and AD with a mobility value almost exactly in the middle of those of the corresponding pure strains show that the two types of A-proteins possess sufficient similarity to be able to form mixed aggregates. The greater spreading of this gradient as compared to AV + AD (Fig. 2a and 2b) is a natural consequence of the fact that the difference in mobilities between AD and AF is greater than that between AV and AD. Therefore, even if the degree of heterogenity in composition remains the same, the mobility-value of the mixed Fey | OV ’ D+F sm AA. SS Fig. 2, Ascending-limb electrophoretic patterns of TMV-pro- tein mixtures at pH 4.9. m=main peak, s~second peak, other abbreviations as in Fig. 1. For explanation see text. 5S. SARKAR aggregates will be scattered over a wider range in case of AF + AD, In case of AV + AF, although two separate peaks are seen (fig. 2c), it is found that the main peak corresponding to protein cylinders has a mobility intermediate between the main AV and the main AF aggregates while the second peak corresponding to discs lies also intermediate between the correspond- ing second peaks of the pure aggregates (Fig. 3). b<—ascending—>| |«—descending—> iar « = £ OO “| mq sti rl — 5 880 |-\— - _ Mm 070 . ce _In. $ & 960 D pey V yor F Feo ® pey V yor F rep Fig. 3. Diagrammatic representation of the relative positions of schlieren peaks produced by TMV-proteins both alone and in binary mixtures at py 4.9, after an arbitrary but equal time of electrophoresis under identical conditions. Abbreviations as in figs. 1 and 2. For explanation see text. It is, however, interesting to note that whenever 4D is present, the united aggregates move as a single boundary. The tendency of AD-units to unite to long cylinders seems to be so strong that the free existence of AV and AF discs (cf, Kramer and Wirrmann, 1958%) is rendered difficult. In any case, the absence of peaks corresponding to pure AF and pure AD aggregates and very probably also of pure AV aggregates in the mixed solutions at pu 4.9 speak strongly for the existence of mixed aggregates, The turbidity observed in each mixed solution at Pu 4.9 was as far as possible removed by centrifug- ing at about 7000 g for 30 minutes in the cold and the opalescent supernatant was examined electro- phoretically. The peaks observed maintained the same contour and mobility as before. After the run, INTERACTION AND MIXED AGGREGATION OF PROTEINS each solution was dialysed against glycine-NaOH buffer, py 10.4 to split the mixed aggregates again into their constituent A-proteins as described be- fore. Electrophoresis in Michaelis buffer py 8.0 exhibited again the presence of two peaks of ap- proximately equal concentrations as judged by their area; the mobilities have already been given in Table 3. In sharp contrast to the formation of mixed ag- gregates among vulgare, flavum and dahlemense proteins, those of vulgare and Holmes’ rib grass strain maintained their identities even in intimate mixture. As the py value of a mixture of AV and AHR was lowered stepwise (sample 4a in Tab. 1), the AHR units aggregated into rods early. At pu 9-9 there are at first three gradients with mobili- ties 0.39, 0.75 and 1.10, which correspond to vul- gare A-protein, aggregated HR protein and aggre- gated vulgare protein respectively (Table 5). After about 14hours of standing, the slowest peak cor- responding to free AV practically disappeared, the fastest assumed a larger area showing the forma- tion of more vulgare-aggregates, while the middle one (HR-aggregate) remained unchanged (Fig. 4). On further lowering the py value to 5.0 the solution became too turbid for electrophoretic runs. | U = (u/sec)/(V/em) i | AV | AHR 8.0 | 0.46 0.85 6.5 | 0.46 0.86 6.1 0.45 : 0.85 5.5 | 0.39 and 1.10* 0.75% 5.5 0.40 and 1.14* 0.79* 5.0 too turbid | | Table 5. Anodic mobilities (U) of observed peaks in a mix- ture of AV and AHR in Michaelis buffer of different pH values. J'=0.037. (Results of sample 4 a from table 1.) * Aggregated. We Fig. 4. Ascending-limb electrophoretic patterns at pH 5.5 showing the gradual transformation of vulgare protein (V) into vulgare-aggregates (Vn) of higher mobility in the pre- sence of aggregated Holmes’ rib grass protein (HRn). The lower pattern was observed 14 hours after the first run (upper diagram). For explanation see text. 783 Obviously, no mixed aggregates were formed. During such a slow and stepwise lowering of the py, the aggregation of HR-protein is complete before vulgare-protein approaches its isoelectric point. To bring about quick aggregation, therefore, a mixture of AV and AHR at py 8.5 (sample 4b of table 1) was dialysed directly against Michaelis buffer of py 5.2 of ionic strength 0.037. On analysis, two sharp peaks were obtained in both arms of the Tiselius cell with mobilities corresponding to pure vulgare- and pure HR-aggregates (Fig. 5 and Table 6), the deviation being well within the range of experimental errors. Both the gradients are sym- metrical and the Schlieren pattern returns to the base line between peaks indicating the absence of mixed gradients having intermediate mobilities. +t Fig. 5. Ascending-limb electrophoretic pattern of a mixture of vulgare- and Holmes’ rib grass proteins at prj 5.2. U = (u/sec)/(V/em) Sample wo ee pooccwnsae= ascending descending AHR alone 0.67 0.65 AV | alone 1.09 | 1.06 AV+t AHR 0.67 and 1.08 0.68 and 1.05 Table 6. Anodic mobilities of AV and AHR at py 5.2. III. Discussion The protein mixtures between vulgare, flavum and dahlemense in weakly alkaline media undergo varying degrees of interaction. Among the various types of protein-protein interactions, a simple dis- sociation and association of peptide chains has been reported from time to time, whereby either relati- vely stable complexes are formed or the different forms remain in a state of dynamic equilibrium. If the six polypeptide chains of .AV and AF could dissociate freely and combine reciprocally with one another, the different mixed units would possess characteristic mobilities as listed below (Table 7), considering that the preferred size of the aggregates remains at six-polypeptide chains and that in such smal] aggregates all the charged groups of each type of polypeptide chain are electrokinetically active. 784 . | Anodic mobility Configuration U == (u/sec)/(V/om) a) 6AV 0.460 b)SAV +1AF 0.445 c)4 AV +2AF 0.430 d)3AV+3AF 0.415 e)2AV +4 4F 0.400 fh lAV+5AF 0.385 g)64F 0.370 Table 7. Calculated mobilities of various mixed aggregates of vulgare and flavum proteins at py 8.0. There are only two observed peaks with mobili- ties which correspond to b and f. It is difficult to visualise why only these two configurations should accumulate preferentially. The mobility values of AV +AD and AF +AD mixtures do not conform well to such 5:1 combinations and the electro- phoretic diagrams speak more for a dynamic equi- librium. Continuously interacting protein mixtures cause boundary anomalies in sedimentation and electro- phoretic studies, which have been summarised and classified on the basis of the velocity constants of the forward and reverse reactions by Atperty and Marvin (1950) '®, Lonesworta (1959)18 and Brown and Timasnerr (1959)! among others, Fretp and Ocsron (1955)?° using human hemoglobin showed that the spreading of a boundary will be greater as the difference of velocities between the forms and their mean lives are greater. These boundary ano- malies should not, however, be confused with the more common types of anomalies encountered with high protein concentrations in comparatively low ionic strengths of the buffer as pointed out by various workers (Svensson, 1944 2!), Jounston and Oasron, 1946 7*, Loncswortu, 1947 23, and ALBERTy, 1948 24). The non-existence of such factors under the conditions of the present investigations is cor- roborated by the absence of boundary anomalies with AV + AHR mixture and by the constancy of mobilities over relatively wide ranges of protein concentrations. An understanding of the dynamic state of equi- librium between the protein mixtures under con- 8 L.G. Loxeswonts, in: Electrophoresis, edited by Milan Bier, Academic Press 1959, Pp. 91 —136. 19 R.A. Brown and S.N. Timasnerr, in: Electrophoresis, edited by Milan Bier, Academic Press 1959. Pp. 317 — 367. 0 E. O. Fintp and A. G. Oesroy, Biochem. J. 60, 661 [1955]. 21H. Svensson, Ark. Kem., Mineralog. Geol., Ser. A 17, 14 f1944]. S. SARKAR sideration demands a knowledge of the mode and rate of dissociation of the proteins. This is difficult, since —as already stated in introduction — the size of an aggregate depends upon various interacting factors. Ultracentrifugal studies on dissociation of TMV-protein into single polypeptide chains have not provided definite clue as to whether the dissociation proceeds through definite steps or not (cf. ANSEVIN and Laurrer, 1959°%, Wirrmann, 195919), With- out, therefore, attempting to ascribe definite mobi- lity values for particular mixed aggregates in weakly alkaline media by postulating particular dis- sociation types as 6P = 3P+3P, 6P24P+2P or 6P 25 P+1P where P stands for a single poly- peptide chain, it can be stated that wherever mixed aggregates of transient existence are formed, it is evidenced by a flattening of the electrophoretic dia- grams and by an approaching together of the respec- tive Schlieren peaks. A simple case would be the one where a vulgare “A-protein’”-unit associates with one of flavum, thus forming shortlived “double- A-protein-units” of intermediate mobility. The marked deviation from enantiography in the two arms of the Tiselius cell is characteristic of reaction-boundaries. The descending boundaries could not be used to calculate exact mobilities for evident reasons but the ascending values were well reproducible. According to Svensson (1946)?5 “the generalisation that all disturbances are smaller on the descending side is no doubt erroneous and the omission of data from the ascending side involves an unjustifiable waste of experimental material”. In contrast to the mobility-values of AV + AF mix- ture at py 8.0 where both schlieren peaks appear to approach each other, it is remarkable that with AD + AF mixture the mobility of the faster peak corresponds very closely to that of pure AD while there is no peak corresponding to pure AF on the ascending side. The difference in mobility between AD and AF is rather large and, therefore, on the ascending side, where the particles move out of the solution into the buffer above, some of the faster units will have a chance to escape the zone of rapid dissociation and reciprocal association. Once out of 2 2 J.P. Jounstox and A. G. Ocsron, Trans. Faraday Soc. 42, 789 [1946]. 3 L. G. Lonasworru, J. physic. Chem. 51, 171 [1947]. °4 R.A. Avserty, J. Amer. Chem. Soc. 70, 1675 [1948]. ®5 H. Svensson, Ark. Kem., Mineralog. Geol, Ser. A 22, 10 [1946]. INTERACTION AND MIXED AGGREGATION OF PROTEINS the zone, the faster particles can gain their own mobility and thereby the front end of the schlie- ren diagram can move with a mobility equal to that of the pure faster component. The situation in the descending arm would be the reverse. On this side there is actually a small but clearly isolated peak corresponding to pure AF-units with a mobility 0.33 to 0.35 (Fig. 6). ee <_—— Fig. 6. Descending-limb electrophoretic pattern of a mixture of flavum- and dahlemense-proteins at pH 8.0. Arrow indicates the direction of migration. The proteins of vulgare and Holmes’ rib grass strains do not obviously exchange their polypeptide chains with each other, though each of them is sub- ject to reversible dissoziation. Both in weakly alka- line media and at py ~ 5 the observed peaks main- tain the characteristic mobilities of pure vulgare- and pure Holmes’ rib grass-proteins. An explanation for the formation of mixed aggre- gates on lowering the py value of binary mixtures of AV, AF and AD described before is superfluous on the basis of the interactions observed in weakly alkaline media. Obviously, with an increase of the H-ion concentration, large aggregates are formed in which polypeptide chains of different strains are packed together in various possible combinations. On simple statistical grounds, particles of greater heterogenity will be more frequent than relatively homogeneous aggregates and thereby the observed peak will depict an intermediate mobility. Somewhat similar observations with hemocyanins from three different species of snails were reported by TiseExius and Horsratz (1939) 76, Asymmetric recombina- tion of different types of human and canine hemo- globins, on rapid neutralisation of a mixture of acid-split half-molecules has been reported by Rosinson and I'rano (1960) 7", Although mixed aggregation between proteins from vulgare, flavum and dahlemense strains of TMV have been observed, no such association was found between proteins of vulgare and Holmes’ rib 26 A. Tisetivs and F.L. Horsrau, J. exp. Medicine 69, 83 [1939]. 27 FE, Rostnson and H. A.Irano, Nature [London] 185, 547 [1960]. 785 grass strains. The polypeptide chains of the latter, therefore, do not fit with those of the wild strain, vulgare. The conditions necessary for an orderly association are similarity or reciprocity in spatial configuration of the polypeptide chains as well as the number and mode of distribution of charged groups on them. The secondary and tertiary struc- tures are certainly dependent on the primary struc- ture, but hardly anything is known as to the maxi- mum permissible variations in the composition and sequence of aminoacids in different mutants in order to just allow a mixed aggregation. In spite of clear-cut differences in the aminoacid composi- tion® and total number of charged groups per poly- peptide chain, the three TMV strains, vulgare, fla- vum and dahlemense, possess sufficient structural similarity for mixed aggregation. In weakly alka- line media AV possesses two negative charges more than AF, while AD has still two more negative char- ges. However, the difference in total charge between AHR and AVP is even greater than that between AF and AD. This alone might prevent mixed aggrega- tion but, as already mentioned, analytical results have demonstrated also radical qualitative and quantitative differences in amino acid composition of the Holmes’ rib grass strain as compared to the other three. At the moment, therefore, it is not possible to differentiate between the roles played by major structural differences and differences in number and distribution of charged groups in the process of mixed aggregation between the TMV-pro- teins. Even then, it is interesting that in reconstitu- tion-experiments the Holmes’ rib grass protein can be successfully used with vulgare-nucleic acid (FRAENKEL-Conrat and Sincgr, 1959 ?8), From the results presented above, two questions easily crop up; viz. 1) do such mixed proteins origi- nate in a living host cell, and 2) what sort of “phy- logenetic” relationships can be assumed between the four strains of TMV used here? If unambiguous experimental proof for simultaneous multiplication of two strains of a virus in one and the same host cell could be available, a treatment of the first question might enter the scope of experimentation. As to the second question, the three strains vulgare, 28 H, Fragnxer-Conrar and B.Sincer, Biochim. biophysica Acta [Amsterdam] 33, 359 [1959]. 786 flavum and dahlemense are certainly “related“ to one another, particularly because flavum is a mutant of vulgare. Holmes’ rib grass strain seems to stand quite apart. At the present moment any statement regarding possible relations in the history of origin of the Holmes’ rib grass and vulgare would involve only speculations. S. SARKAR I am grateful to Professor G. Metcuers for allowing me to carry on the investigations in the Max-Planck- Institute for Biology and for his constant interest and valuable criticism. My thanks are also due to Dr. E. Kramer for his continuous helpful directions and tech- nical supervision. My stay abroad was financed by a fellowship of the Max-Planck-Gesellschaft, for which I am very thankful.