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When tobacco mosaic virus (TMV) protein is polymerized at pH values above 7 in unbuffered solutions, either by raising temperature at constant ionic strength or by increasing ionic strength at constant temperature, a 20 S component is formed having bound only the very small amount of H+ ion supplied by the unpolymerized protein. When hydrogen ion is added by titration during polymerization so as to keep pH constant, as would occur automatically if a buffer were present, a 20 S component is formed with one H+ ion bound each for half of the subunits. Thus, a 20 S form with and a 20 S form without bound H+ ion exist. Furthermore, the 20 S form without bound H+ ion binds H+ ion when supplied by titration to produce a 20 S form with the same amount of bound H+ ion as when H+ ion is supplied during the polymerization.

Calcium ion titrations were performed on solutions of tobacco mosaic virus coat protein using a calcium-specific ion-exchange electrode. Isolated coat protein was found incapable of binding calcium ions under equilibrium conditions at pH values above its iso-ionic point (pH 4.3 to 4.6). However, calcium ions were found to bind to coat protein under non-equilibrium conditions, which suggests that the isolated coat protein has the proper conformation to bind calcium ions at the iso-ionic point.

The effect of the dipolar ions, glycine, glycylglycine, and glycylglycylglycine on the polymerization of tobacco mosaic virus (TMV) protein has been studied by the methods of light scattering and ultracentrifugation. All three dipolar ions promote polymerization. The major reaction in the early stage is transition from the 4 S to the 20 S state. As in the absence of dipolar ions, the polymerization is enhanced by an increase in temperature; it is endothermic and therefore entropy-driven. The effect of the dipolar ions can be understood in terms of their action as salting-out agents; they increase the activity coefficient of TMV A protein, the 4 S material, and thus shift the equilibrium toward the 20 S state. The salting-out constants, K, for the reaction in 0.10 ionic strength phosphate buffer at pH 6.7 was found by the light scattering method to be 1.6 for glycine, 2.5 for glycylglycine, and 2.5 for glycylglycylglycine. A value of 2.7 was obtained by the ultracentrifugation method for glycylglycine in phosphate buffer at 0.1 ionic strength and pH 6.8 at 10 degrees C. For both glycine and glycylglycine, K increases when the ionic strength of the phosphate buffer is decreased. This result suggests that electrolytes decrease the activity coefficient of the dipolar ions, a salting-in phenomenon. However, the salting-in constants evaluated from these results are substantially higher than those previously determined by solubility measurements. The effect of glycine and glycylglycine on polymerization was studied at pH values between 6.2 and 6.8. The effectiveness of both dipolar ions is approximately 50% greater at pH 6.8 than at pH 6.2. The variation of the extent of polymerization with pH in the presence of the dipolar ions is consistent with the interpretation that approximately one hydrogen ion is bound for half of the polypeptide units in the polymerized A protein.

Calcium and potassium ion titration experiments were performed on solutions of tobacco mosaic virus RNA using ion-specific electrodes. The data obtained were analyzed using Scatchard and Klotz plots for the number of binding sites per nucleotide (n), and the apparent stability constant for complex formation, beta Me. The experimental design also allowed for the determination of the number of protons released per metal ion bound, chi. The calcium ion titration in water yielded values of 0.45 for n, 6.03 for log beta Ca and 0.24 for chi. When this titration was repeated in 0.01 M-KCl, the values were found to be 0.11 for n, 5.08 for log beta Ca and zero for chi. An aqueous potassium titration was also performed, with values for n, log beta K and chi of 0.25, 2.96 and less than 0.10, respectively.

The effects of temperature (T), ionic strength (mu), and pH on the polymerization of the coat protein of the E66 strain of tobacco mosaic virus (TMV) from the 4 S form (A), a trimer of the polypeptide chain, to the 20 S form (D) were investigated by the method of sedimentation velocity. Interpretations of thermodynamic parameters were based on only those data obtained in experiments for which reversibility could be demonstrated both by lowering temperature and by lowering concentration. E66 protein differed from vulgare TMV in that, in position 140, lysine replaced asparagine. Thus, E66 protein should be less hydrophobic than vulgare protein and K's, the salting-out constant, should be less. The charge on unpolymerized E66 protein was -3 proton units per polypeptide chain, compared to -4 for vulgare protein. The electrical work contribution, delta W*el, for E66 protein should be (-3/-4)2, or 0.5625 that for vulgare. The results were that delta W*el at pH 6.7, 15 degrees C, and mu = 0.1 was 0.700 kcal/mol for E66 protein compared to 1.22 for vulgare. The experimental ratio was 0.574; K's = 2.16 for E66 compared to 4.93 for vulgare. Hydrogen ions (1.5) were bound per A unit, or 0.5 per polypeptide chain, in the formation of D from A. delta H*, the enthalpy change per mole of A, was 33 kcal at pH 6.7 and 36 at pH 6.9, compared to 30 at both pH values for vulgare protein. delta S*, the entropy change per mole of A, was +132.1 e.u. for E66 compared to 127.4 for vulgare. Entropy-driven processes are found in dynamic biological situations. Ready reversibility at biological temperatures is a requirement, yet the polymer structures must be strong and well ordered. This is achieved through a large number of weak bonds between subunits, combined with ready reversibility under slightly changed conditions. The significant role of water is to facilitate depolymerization by binding to subunits.

Calcium ion titrations were performed on solutions of tobacco mosaic virus using a calcium-specific ion-exchange electrode. Scatchard analyses were used to obtain the number of calcium ion binding sites per protein subunit (n) and the apparent stability constant for complex formation (beta' Ca). These experiments were performed on unbuffered solutions, in either water or 0.01 M-KCl, to allow a determination of the number of hydrogen ions released per calcium ion bound (chi). The results indicate that near neutrality, the virus particle possesses two calcium ion binding sites per subunit having apparent stability constants greater than 10(4) M-1. The results are interpreted as if these two sites are non-identical and titrate independently. The higher affinity site for the virus in water has a value of log beta' Ca, which varies from about 8.5 at pH 8.5 to about 3.9 at pH 5.0, and for the virus in 0.01 M-KCl has a value that varies from about 6.2 at pH 8.0 to about 3.7 at pH 5.5. The higher affinity site for the virus in water binds up to two competing hydrogen ions, one with an apparent pKH value greater than 8.5 and the other with a value that varies from 6.0 at pH 5.5 to 7.3 at pH 8.0. For the virus in 0.01 M-KCl, only the competing hydrogen ion binding with an apparent pKH value greater than 8.5 remains. The results could be interpreted as indicating that the electrical charge on the virus particle has a constant value in the pH range 5.5 to 8.0 despite the fact that hydrogen ion titration curves for the intact virus particle indicate that the charge should vary from about -1 per subunit at pH 5.5 to about -4 at pH 8.0.

Holmes ribgrass virus (HRV), because of serological results, is regarded as a distantly related strain of tobacco mosaic virus (TMV). HRV protein differs substantially in amino acid sequence from TMV protein, especially in that it contains one histidine residue and three methionine residues, compared to none of either for TMV protein. Ultracentrifugation and hydrogen ion titration data on HRV protein, similar to those obtained previously for the early stage polymerization of TMV and E66 proteins, demonstrated some similarities and more distinct differences from those of the other two proteins. The major similarities are that the early polymerization of HRV protein is entropy driven and the first major polymerized product is a 20 S component, presumably a double disk or two-turn helix, as in the case of the other proteins. The major differences are that the unpolymerized HRV protein sediments at 3 S rather than at the 4 S for the others; it is presumably a dimer of the polypeptide chain. The enthalpy of polymerization per mole of A protein, delta H*, is 18,400 cal for HRV protein, compared to about 30,000 for TMV protein. One mol of H+ ion/mol HRV A protein, compared to 1.5 for TMV and E66 proteins, is bound during polymerization to the 20 S state. Contrasted with the other proteins, very little if any electrical work contribution was detected for the HRV protein. A major difference was found in hydrogen ion titration. Unpolymerized HRV protein binds hydrogen ions significantly in the unpolymerized A protein state, unlike the A proteins from the other two viruses.

The endothermic polymerization of the coat protein of tobacco mosaic virus has been studied by high-sensitivity differential scanning calorimetry, with control experiments involving turbidimetry and sedimentation velocity measurements. The variation of the apparent extent of polymerization under conditions close to equilibrium as the temperature is raised follows a course which is difficult to duplicate on the basis of simple models for the process. The enthalpy of polymerization at low protein concentration varies from 12.5 kcal (mol of monomer)-1 (17500 daltons) under conditions where the product is largely a mixture of short helical rods to 6.0 kcal ol-1 for the formation of double disks containing 34 monomer units. In the former case, the polymerization is accompanied by a decrease in apparent heat capacity of 350 cal K-1 mol-1 while in the latter there is an increase of 150 cal K-1 mol-1. These results constitute evidence that these two types of polymerization involve intersubunit bonds of quite different chemical character.

Crude preparations of PR8 influenza virus, obtained by high-speed centrifugation, contain two electrophoretically distinct components. One of these, present to the extent of 10 to 20 per cent, was identified by electrophoresis, centrifuge, and activity tests, as an impurity similar to or identical with a high molecular weight acidic substance shown by Knight to be elaborated by normal uninfected embryos. The other component, present to the extent of 80 to 90 per cent, appeared to represent the active virus. The virus fraction was separated from the impurity by repeated fractional centrifugation. It then appeared homogeneous in the analytical centrifuge and in the Tiselius apparatus, and possessed an isoelectric point at pH 5.3 as measured by the micro-electrophoresis method.

A study has been made to establish the statistical significance of results obtained in mouse infectivity titrations of influenza virus. Five titrations, each composed of five replicas, were carried out and 50 per cent end points were calculated for each titration. Three criteria for evaluating the end points were employed, namely, the presence or absence of pulmonary lesions, the occurrence of death, and a weighted composite taking into account both the extent of lung consolidation and the occurrence of death. Standard deviations of the distribution of end points obtained by each method were computed, and from these data levels of probabilities for significance in the differences between end points were determined. It was found that the chances are 19 out of 20 that differences of 0.99, 0.77, and 0.73 logarithmic units, respectively, for the lesion, the death, and the weighted end points are significant.

The biophysical properties of several preparations of PR8 influenza virus have been studied. Electron micrographs showed slightly irregular, circular particles with an average diameter of 115 mmicro and a standard deviation of the distribution of diameters of 15 per cent. The specific volume was determined with a pycnometer to be 0.79. The sedimentation rate was found to vary inversely with the concentration of virus. The extrapolated value for one preparation was 722 Svedberg units and the value for another preparation was 658 units. Sedimentation studies in sucrose solutions of varying densities showed a non-linear dependence of sedimentation rate upon solvent density, indicating that the density in solution increases with increasing sugar concentration. The virus particles floated in a sugar solution with a density of 1.18. The density in the absence of sucrose was estimated to be about 1.1. The virus was shown not to lose infectivity very rapidly in sucrose solutions. The spreading of the boundary during sedimentation was shown to be represented by a standard deviation of the sedimentation rate equal to 8 per cent of the mean. This corresponds to a distribution of diameters with a standard deviation of 4 per cent of the mean. A more slowly sedimenting component was observed with the ultracentrifuge in the highest concentrations of several of the preparations. The intrinsic viscosities, calculated on the basis of concentrations expressed as grams per cubic centimeter, of several highly purified preparations were determined to be between 11.3 and 16.5. The variation of sedimentation rate with concentration was shown to be due entirely to the variation of solution viscosity with concentration. The viscosity of the virus preparations can be explained as being due in great part to the presence of the slower sedimenting contaminating components which possess a very high intrinsic viscosity. The average particle diameter of the anhydrous PR8 virus particle was calculated from sedimentation and specific volume data to be about 80 mmicro. The discrepancy between this value and that obtained from the electron micrographs, the greater size distribution from the electron micrographs, the slight irregularities in some of the particles as observed in the electron micrographs, the behavior of the sedimentation process in sucrose solutions of different densities, and the inactivation of the virus by withdrawal of electrolytes can all be explained in a straightforward manner if it is assumed that the virus particles in solution contain, in addition to the constituents shown by chemical analysis, about 60 per cent by weight of water. It is estimated that such hydrated virus particles are about 100 mmicro in diameter.