SOME PHYSICOCHEMICAL PROPERTIES OF SPECIFIC POLYSACCHARIDES* By MICHAEL HEIDELBERGER anp FORREST E. KENDALL (From the Department of Medicine, Presbyterian Hospital and College of Physicians and Surgeons, New York) (Received for publication, March 27, 1931) In recent years the study of immunologically specific poly- saccharides has not only contributed to the understanding of bacterial specificity (1) but has also proved of purely chemical interest, because of the unusual structure of certain members of the group. Thus the specific polysaccharide of Type III pneumo- coccus has been shown to be a polyaldobionic acid built up of glycurono-glucose units (2, 3). It is, moreover, a typical colloid in the sense that it does not diffuse through collodion or parchment into water, and is, by virtue of its structure, so strong an acid that its aqueous solutions turn Congo red paper blue. Hence it seemed of interest to study some of the physicochemical properties of solu- tions of its salts, the more so as little is known of the behavior to be expected of an organic, colloidal, strong electrolyte devoid of basic groups. Accordingly, the sodium salt of the Type III pneu- mococcus specific polysaccharide was subjected to a study of its viscosity, conductance, and its behavior on diffusion. Measure- ments were also made on solutions of other specific polysaccharides. The diffusion data are reserved for a forthcoming paper dealing with the molecular weight of specific polysaccharides, in which connection the data recorded in the present communication will again be taken up. Physicochemical studies have previously been made of gum arabic, a carbohydrate of a character somewhat analogous to that of the Type IIT pneumococeus polysaccharide. This substance, however, is the salt of a much weaker acid than the Type III * The work described in this communication was carried out under the Harkness Research Fund of the Presbyterian Hospital, New York. 127 128 Specific Polysaccharides pneumococcus carbohydrate, since the equivalent weight of arabic acid is about 1200, while the Type III substance (hereinafter referred to as 8 III) possesses one carboxy! group for every 340 of molecular weight. Thomas and Murray (4) have given a titration curve of arabic acid and also studied its osmotic pressure and vis- cosity at various hydrogen ion concentrations, and concluded that its behavior might be satisfactorily explained on the basis of the 100 98 Equivalent conductance A (corrected for water) 0 1 2 4 5 6 7 3 1001T- Fig, 1. Conductance of sodium salt of pneumococeus Type IIT specific polysaccharide. Donnan equilibrium. The application of this conclusion to the viscosity data was rejected by Taft and Malm (5) on the basis of an extended study which included conductivity measurements. These resulted in the conclusion that the current was carried mainly by the inorganic ions present. TABLE I Conductivity of Na Salt of S III at 25° + 0.008° Total weight Equivalents} Volume (at Resietance 10 10 oer eran oer Equivalent of stock solu: | "x 108 25°) ox“ paging | OR RY | ductance (L’) [rected for water: conductance gm. ml. equivalents ohms mhos mhos mhos 0. 0 929.7 | 0 0 9917.0 | 1.00837 | 0.01018 | 0.005996 1.0631 3.8862 930.7 | 0.041755 | 0.647 9321.5 | 1.07279 | 0.07460 | 0.043936 0.037940 90.863 10.6849 39.059 940.4 | 0.41534 2.04 6286.6 | 1.59069 | 0.59250 | 0.34895 0.34295 82.571 20.2357 73.972 949.9 | 0.77873 2.79 4859.0 | 2.05804 | 1.0599 0.62420 0.61820 79.385 35.9751 | 131.507 965.4 | 1.3622 3.69 3597.8 | 2.77948 | 1.7813 1.0491 1.0431 76.575 66.4993: | 243.088 995.6 | 2.4416 4.94 2460.8 | 4.06872 | 3.0655 1.8054 1.7994 73.697 105.4481 | 385.466 1034.2 | 3.7272 6.11 1805.0 | 5.54017 | 4.5420 2.6750 2.6690 71.609 equivalents Concentration of stock solution, 3.6555 X 10°’ ————-—.. gm. solution in air Equivalent weight, 340. The cell was shunted by a resistance coil (S) of 10018.1 ohms in the measurements, R, bridge reading. R’, actual resistance of cell. K, cell constant = 0.58895. 1 1 1 1000 L == 1 Bi L=L! —Iggih =a Ro RS 7 R? Tepueyy “TW pue sesreqieprey “WH 621 130 Specific Polysaccharides EXPERIMENTAL Conductivity Determinations—The conductivity data were ob- tained with the arrangement of apparatus described by Shedlovsky (6). An S III solution was added to a new type of cell (7) from a TABLE 11 Viscosities of S III Measured with Bingham Viscometer, No. 128, K = 1.99 X 10-5 2 | & = #| Solvent j Time g 4 2 isp B |i my gym gp | eye & | 8 a 7 °C. | per cent Gree | see. | Settee | Sotaes 20 (2.93 H.0 188.90,6859 [25.78 |1.0050/25.65 |24.65 | 8.41 0.293 “ 117 .39}1890.6) 3.248/1.0050; 3.232; 2.232) 7.62 0.293 “ 216.4 | 752.4) 3.234/1.0050; 3.218] 2.218) 7.57 0.0293) 214.05] 352.7) 1.503/1.0050, 1.495) 0. 495/16 .89 0.0293) 99.18) 799.7) 1.539/1.0050) 1.531) 0.531/18.12 0.0293) “ 280.3 | 266.1) 1.484]1.0050] 1.477) 0.477/16.28 30 10.0293) “ 99.12} 617.9] 1.2190.8007| 1.522; 0.522/17.82 0.0293; “ 208.99) 286.4] 1.191/0.8007] 1.488) 0.488/16.66 0.0293) “ 280.32! 211.3] 1.179}0.8007| 1.472; 0.472/16.11 20 (0.0293) 0.17 m NaCl /280.04| 192.8] 1.074!1.0090] 1.064) 0.064] 2.18 0.0293) 0.17 =“ 193.23} 278.7) 1.072/1.0090| 1.062) 0.062) 2.12 0.0293) 0.17‘ “ 110.87| 487.0) 1.075]1.0090) 1.065) 0.065) 2.22 36 (0.0293) 0.17 ‘* “ 111.08] 388.3) 0.858/0.809 | 1.061; 0.061) 2.08 0.0293} 0.17 ‘* “ |212.37) 202.8] 0.857|0.809 | 1.059; 0.059] 2.01 20 |0.0586} 0.17 ‘* “ |233.86) 243.7] 1.134/1.009 | 1.124) 0.124) 2.12 0.0586, 0.17 132.31} 431.4] 1.134/1.009 | 1.124) 0.124) 2.12 0.0293] 0.85 ‘* 132.25] 426.9] 1.1241.078 | 1.042) 0.042) 1.43 0.0293) 0.85 * 166.79) 338.4 1.123)1.078 1.042] 0.042] 1.43 0.0293) 0.85‘ “ 219.27) 257.8 1.124.078 1.043) 0.043] 1.47 weight burette, so that increasing concentrations were measured without emptying the cell. The equivalent weight was taken as 340 and measurements were made in the range 0.039 to 3.85 milli- The extrapolation to limiting conductance was made with the aid of a derived slope curve by use of the formula given in Fig. 1. That the results were not influenced by hydrolysis is indicated by equivalents. The data are recorded in Table I and Fig. 1. M. Heidelberger and F. E. Kendall 131 che fact that no shift in pH could be observed with either methyl red or phenol red as indicator on diluting a 30 mm solution 100 times with freshly boiled distilled water. TABLE Viscosities of S IIT Solutions. IIl Ostwald Viscometer at 20° Substance Solvent en Time percent | sec. H,0 92.6 STII H.0 0.293 |303* “ 0.0586 {174.8 7 0.0293 {147.9 “ 0.0195 {187.1 “ 0.01465)132.5 “6 0.01172/124 .4 KCl H,0 1.21 u | 86.6 8 ITI 1.21 m KCl 0.2930 |129.9 121° « 0.0293 | 90.5 KCl H,0 3.35 M | 82.4 S III 3.35 m KCl 0.293 | 99.4 3.85‘ 0.0293 | 84.3 Na.HPO, H,O 0.05 mw | 95.5 8 ITT 0.05m Na:HPO,| 1.00 (406. 0.05 “ “ 0.10 /117.0 HCl H.0 0.27 m| 94.1 8 III 0.27 m HCl 0.0293 | 97.7 HCl H.0 2.60 m {101.5 $ III 2.60 m HCl 0.0293 |106.1 NaCl H,0 0.17 uw | 93.7 § Ill 0.17 m NaCl 0.0293 | 99.6 Time, sol- vent séc. SSSESS Go > Oe Mh HD OD 95.5/4. 95.5)1. 94.1 101.5 93.7 (al al aaa ae eSESSSN 1.04 1.05) 1.06 Nep eosooon ReSSEN 0.04 0.05 0.06 "ep Cc 7.75 15.20 20.48 24.62 29.35 29.10 Se oo 99 GO 90 ©? we BARS SS "ep O corrected for vol- ume of KC! 1.65 0.64 * Corrected for relative density of solution, a = 1.003; Tors. X d. The conductivity determinations were made at The Rockefeller Institute for Medical Research by Dr. T. Shedlovsky, and it is a pleasure for the writers to express their indebtedness to him for his freely given time and invaluable aid in the interpretation of the 132 data. Specific Polysaccharides Thanks are also due Dr. Dunean A. MacInnes of the Rocke- feller Institute for his interest and assistance. TABLE IV Viscosities of S I and S II in Ostwald Viscometer at 20° - , i %, Subs, | sotvene | onsen | crime | me] me | mee | WP | Oe per cent sec, see, H,0 92.9 SI H.0 0.3273 |187.9*} 92.9 | 2.05 | 1.05 | 3.21 | 1.84 “ 0.06546|122.3 | 92.9 | 1.32 | 0.32 | 4.89} 1.25 “ 0.03273)108.7 | 92.9 | 1.17 | 0.17 | 5.20] 0.94 “ 0.02181/105.6 | 92.9 | 1.14] 0.14 | 6.42 | 0.95 “ 0.01637/103.2 | 92.9 | 1.11 | 0.11 | 6.72.1 0.86 . 0.0131 |100.5 | 92.9 | 1.08 | 0.08 | 6.11 | 0.70 P ¢c KCl H.0 1.34 m| 86.0 meta ‘or vol- ume of KCl SI 1.34 m KCl 0.3273 {116.5 | 86.0 | 1.35 | 0.35 | 1.07 | 1.03 3.35‘ “ 0.3273 |109.6 | 82.4f| 1.33 | 0.33 | 1.01 | 0.90 0.17 NaCl | 0.3273 |132.8 | 93.7+/ 1.42 | 0.42 | 1.28 0.27 ‘* HCL 0.3273 1141.4 | 94.1f; 1.50 | 0.50 | 1.53 2.60 “ 0.3273 |153.0 |101.5+/ 1.51 | 0.51 | 1.56 0.008 m HCl | 0.3273 |164.5 | 92.6t) 1.78 | 0.78 | 2.38 0.009“ “ 0.2075 146.2 | 92.6f] 1.58 | 0.58 | 1.95 H.0 93.1 si H,0 1.106 |113.7t] 93.1 | 1.22 | 0.22 | 0.20 “ 0.221 | 97.2 | 93.1 | 1.04 | 0.04 | 0.18 NaCl “ 0.85 | 96.5 SII 0.85 m NaCl | 0.553 /103.2 | 96.5 | 1.07 | 0.07 | 0.13 * Corrected for relative density of solution, a = 1,002. + From Table III. Time for H,O is used for 0.008 and 0.009 m HCl. t Corrected for relative density of solution, d’ = 1.007. Viscosity Measuremenis—The viscosity data given in Table II were determined as described by Bingham (8) with the Bingham viscometer at Lafayette College, and the writers wish to thank Professor E. C. Bingham for extending to them the hospitality of his laboratory and the benefit of his personal interest. All pres- M. Heidelberger and F. E. Kendall 133 sures were corrected for temperature, hydrostatic head, and flow. The viscosities were expressed in absolute units and the relative and specific viscosities calculated. The remaining viscosities were determined with an Ostwald viscometer at 20°, the value obtained with an § III solution within the range of the Hagen-Poiseuille law checking closely with that found with the aid of the Bingham instrument. 5.0 cc. of solution were used in every case. Potassium chloride was used instead of sodium chloride at the higher salt concentrations since its effect on the viscosity of water is almost negligible. TABLE V Viscosities of Specific Polysaccharides in Ostwald Viscometer at 20° Concen- ; Time, ep Substance tration Time a Txd solvent Np "ep a Specific gum arabic (8.G.A.) per cent sec, 8c, H,0 93.1 8.G.A, | 4.85 143.2 | 1,022 | 146.4 | 93.1 | 1.57 | 0.57 | 0.12 “ 0. 485 99.0 | 1.002 | 99.2] 93.1 | 1.07 | 0.07 | 0.14 “ 0.0485 | 94.0 93.1 | 1.01 | 0.01 | 0.21 Bovine tubercle bacillus polysaccharide (B.B.G. 526 I A*) H,O0 92.6 B.B.G. | 1.218 } 102.1 | 1.003 | 102.4; 92.6 | 1.11 | 0.11 | 0.09 “ 0.1218 | 93.9 92.6 | 1.01 | 0.01 | 0.08 * To be described in a later publication. The results are given in Tables III to V and in part in Fig. 2. Titration of Type I Pneumococcus. Specific Polysaccharide—It will be recalled that SI is both a weak base and strong acid (9). Since the isoelectric substance dissolves only slowly in alkali the titration was carried out as follows: A weighed sample of anhydrous SI was wet with water and dissolved with a measured amount of n/14 hydrochloric acid. The solution was then titrated back through the isoelectric point with n/14 sodium hydroxide. The finely divided precipitate dissolved readily at a reaction which was still acid to litmus, and the titration was continued to alkalinity THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL, XCV, NO, 1 134 Specific Polysaccharides to phenolphthalein or thymolphthalein, very little additional alkali being required between the turning points of the two indi- cators. 0.3153 gm. of S 1 58, dissolved in 10.03 ce. of n/14 HCl, required 25.23 cc. of n/14 NaOH (0.15 cc. being deducted as indicator blank) to blue color to thymolphthalein, or 15.20 cc. in excess, equivalent to 1.086 ec. of n NaOH. Acid equivalent, 290. 0.4048 gm. of SI 59 B, dissolved in 10.03 cc. of w/14 HCl, required 27.28 cc. of N/14 NaOH to bright pink to phenolphthalein, 100 200 500 0015 x - AA ao10 Flow|per secondat 20 C] 20 Ns < 10 r p Pre; wper sq.m. } ee Copen T Cancn ——— | 3.27 if } O10 40 on Conte (gmiper rooees Fra. 2. Viscosity relations of sodium salts of specific polysaccharides. Curve I, S III; Curve II, 8 I; Curve III, 8. G. A. (ordinates X 0.1; abscisse X 10); Curves IV and V, S III 0.293 and 0.0293 per cent, respectively. or 17.25 ec. in excess, equivalent to 1.232 cc. of n NaOH. Acid equivalent, 328. Mean acid equivalent, 309. DISCUSSION The high values of the equivalent conductance shown in Table I and Fig. 1 indicate that the sodium salt of S III is a strong elec- trolyte, since A, which is 95.7 at infinite dilution, drops to only 71.6 at a concentration of 3.85 milli-equivalents. M. Heidelberger and F. E. Kendall 135 The conductivity of S III, however, does not follow the linear relationship between A and the square root of the concentration, A = A, — a VC, derived empirically by Kohlrausch and given theoretical significance by Debye and Hiickel and by Onsager (10). In a highly ionized substance, the slope of the conductance curve, a, depends largely on the valence type, increasing with the valence. In the case of an electrolyte which is incompletely dissociated, such as thallium chloride (cf. (10)), dilution decreases the slope, owing to the increase in the number of conductors, and the resulting curve is concave toward the origin. In the STII salt, however, this effect is more than counterbalanced, and the curve becomes convex toward the origin. This may be explained by assuming, with increasing dilution, the formation of ions of higher and higher valence type as additional COONa groups dissociate. The possibility of this taking place is shown by the structural formula for the S III salt given below in another connection. Thus, the slope, a, of the conductivity curve would tend to increase with dilution, rather than decrease, owing to the preponderating influ- ence of the increasing valence. That even a simpler substance may show a. similar deviation from a straight line relationship is readily demonstrated by plotting the equivalent conductance curves calculated from Noyes and Lombard’s measurements for the tetra and penta sodium salts of benzene pentacarboxylic acid (11). Since the limiting conductance of the sodium ion is 50.0, that of the Type III polycarboxylate ion (equivalent weight 340) would be 95.7 — 50.0, or 45.7, indicating that it is a ready carrier of elec- tricity. Its behavior is thus very different from that ascribed to a polysaccharide anion such as that of gum arabic (equivalent weight 1200) by Taft and Malm (5), who consider the inorganic ions responsible for the entire current carried. The value of the limit- ing conductance is, indeed, very close to recent values ascribed to the caseinate (equivalent weight 2000) (12) and globulinate ions (equivalent weight 3000) (13). The S III polyearboxylate ion, however, is free from basic groups and has a carboxyl group for every 340 of molecular weight. Since all of the available evidence indicates that there are at least eight to ten such groups in the molecule, the cumulative effect of the negative charges on the S IIT ion would be very large. That a total charge of this magnitude would result in large interionic or Coulomb forees can readily be appreciated. 136 Specific Polysaccharides Tables II and III show that although the specific viscosity, tp = tr — 1, is independent of a 10° increase in temperature, indi- cating that there is no association or dissociation on changing the TABLE VI Specific Volumes of Dissolved Specific Polysaccharides According to Kunjiiz’s Formula Substance Solvent rte snl ty ? Specific “2 per cent per cent gm. solute SI H:0 0.298 | 3.27 | 23.5 80 “ 0.0586 } 1.89 | 13.4 | 230 “ 0.0293 | 1.60 | 10.0 ; 340 “ 0.0195 | 1.48 8.4 | 430 “ 0.01465] 1.43 7.7 | 530 “ 0.01172) 1.34 6.3 | 540 1.21 m KCl 0.2930 | 1.50 8.7 30 1.21 0.0293 | 1.05 1.1 38 3.35 ** “ 0.2930 | 1.21 4.2 14 3.35 “ 0.0293 ) 1.02 0.44) 15 0.05 ‘* NasHPO, /1.00 4.26 | 28.3 28 0.05 “ “ 0.10 1.23 4.6 46 SI 4:0 0.3273 | 2.05 | 14.9 46 “ 0.0327 | 1.17 3.5 | 110 “ 0.0218 | 1.14 3.0 | 140 “ 0.0131 | 1.08 1.8 | 140 1.34m KCl 0.3273 | 1.35 6.5 20 3.35" “ 0.3273 | 1.33 6.2 i9 SII H.0 1.106 | 1.22 4.4 4 7 0.221 | 1.04 0.87 4 0.85 u NaCl 0.553 | 1.07 1.6 3 8.G.A. H,0 4.85 1.57 9.6 2 “ 0.485 | 1.07 1.6 3 Nabenzene pen-| “ 2.04 1.09 2.0 1.0 | 0.044 tacarboxylate | “ 0.816 | 1.04 0.87 1.1 | 0.049 (Noyes andj} “ 0.245 | 1.015) 0.33 1.3 | 0.061 Lombard) temperature in this range, aqueous solutions of S III obey neither the Hagen-Poiseuille law, nor the Einstein relation, » = 10 ( +K *), from which 7 should equal a constant. It is clear M. Heidelberger and F. E. Kendall 137 (Fig. 2) that the latter function increases to a maximum with in- creasing dilution, just as the equivalent conductance increases with dilution. It is, therefore, possible to ascribe the increasing deviation from the Einstein viscosity relation to the same cause; namely, the ionization of additional COONa groups and the resultant increase in the charge on the polycarboxylate ion. In this instance the relation 2 ig found to hold over a limited range of concentration, but this is not so evident in the other specific polysaccharides studied. Moreover, the viscosity determinations made by Noyes and Lombard (11) on solutions of sodium benzene pentacarboxylate show a 40 per cent increase in 2? with decreasing concentration Cc from 2 per cent (50 mm) to 0.25 per cent (6 mm) (Table VI). Although this is small compared with the effect noted in the case of SILI, the negative charge due to the piling up of carboxy] groups on even a substance of low molecular weight appears to result in a viscosity effect in the same direction. It is also possible to ascribe the observed viscosity effects to hydration of the specific substance. If this be true, the hydration must be of a most unusual magnitude. Otherwise, at the low con- centrations of § IIT at which a is still increasing, the concentration of free water in the system would be practically constant. If hydration be the cause of the observed anomalies, it should be possible to calculate its extent by means of Kunitz’s modification _14+05¢ (14), 9 = G+e) +)? lead to enormous values for the specific volume in dilute aqueous solution (Table VI), 1 gm. of S III being capable, according to the formula, of combining with as much as 540 gm. of water. It is also shown in Fig. 2 that if the rate of flow per second of dilute solutions of 8 ITI in water is plotted against the pressure, a straight line is obtained for each concentration. These lines do not pass through the origin, but intersect the axis at a positive value for P which increases with dilution. Thus a certain pressure must be exceeded before flow starts, and the solution behaves as a plastic solid. This might be accounted for by assuming a definite orien- tation of the highly charged polycarboxylate ion at high dilutions of Einstein’s formula, and this does indeed | | Glucose Glucose Glucose Glucose Glucose [oft fot) —o-| | 0] | ORCOO- ORCOO- ORCOO- ORCOO- ORCOO~ ORCOO- ORCOO- ORCOO- ORCOO- ORCOO- | FO | | }o4| || Glucose Glucose Glucose Glucose Glucose + + 138 Specific Polysaccharides at which dissociation is approaching completion. This would also entail an orientation of the accompanying sodium ions, and a definite structure would result, somewhat as follows: Nat Nat Nat Nat Nat Na* Na Na in which RCOO- represents the glycuronic acid residue in gluco- sidic union with the glucose residues (cf. (3)). Such an arrangement should also result in a maximum value of a and is consistent with the explanation given for the positive deviation from the square root conductivity relation at high dilutions. In the case of the specific polysaccharide of Type I pneumo- coccus the viscosity anomalies are smaller than in the case of § ITI, and this might be ascribed either to the smaller magnitude of the Coulomb forces engendered by the § I anion, or to the lower extent of the hydration. The acid equivalent of § I is somewhat lower than that of S III, but since there is one basic group for approxi- mately every three carboxyl groups, the total negative charge on the S I ion would be less than that of 8 III, assuming the molecular weights to be approximately the same. Also, calculation of the hydration by Kunitz’s formula yields lower values than in the case of § III, so that either explanation might be valid. In the case of SII and the specific gum arabic, with their acid equivalents of approximately 1000, both viscosity and hydration are low. Since there is an apparent parallel between the total acidity of the specific polysaccharides considered and their hydra- tion as calculated by the Kunitz formula, it is possible that the unusual hydration, or at least the unusual viscosity, of the S III and S 1 is conditioned by the unusual magnitude of the total negative charge on these ions. Association of abnormally high viscosities with an acid poly- saccharide has been shown qualitatively by Raistrick and Rintoul (15) in the case of luteic acid, a polysaccharide produced from |= Na re Na M. Heidelberger and F. E. Kendall 139 glucose by Penicillium luteum, Zukal. The structural unit is eomposed of 2 molecules of glucose and 1 of malonic acid, one of the carboxyl groups being free. Moreover, Staudinger and Kohlschiitter (16) have recently continued earlier work by Staudinger and Urech (17), giving preliminary viscosity data on polyacrylic acids. They fix the relative viscosity of a 0.1 equivalent solution of a sodium salt at the enormous value of 114 and the relation a at about 1140, values far higher than obtained in the case of SIII. The polyacrylic salts differ from those of S III, however, in hydrolyzing readily and in the smaller equivalent conductivity of their aqueous solutions. Staudinger originally considered the high viscosity of polyacrylic acid salts to be due to hydration caused by the high ionic charge, but now ascribes it to “swarms” caused by increasing ionization. If, as seems agreed, these high viscosities are conditioned by the piling up of large Coulomb forces, regardless of the mechanism by which this is brought about, a substance such as acrylic acid, with an acid equivalent of only 86 would be expected to develop higher viscosities as the chain of carbon atoms and carboxyl groups is lengthened by polymerization, than would the aldobionic acid structural unit of S ITI, with its acid equivalent of 340. Tables II and II] show clearly the effect of electrolytes in dimin- ishing the viscosity (and in Table VI the hydration) of the solutions of S III and in bringing them into agreement with the Hagen- Poiseuille law and the Einstein equation. It is shown in Table IV that a increases to a maximum with increasing dilution in the case of the sodium salt of SI as in the Usp VE is constant over a very limited range. Although titration shows that the acid equivalent of SI is lower than that of SITI, the greater number of carboxyl groups in the molecule and their expected effect on the total charge and viscosity appear to be more than negatived by the simultaneous presence of basic groups. However, the internal compensation of Coulomb forces is insuffi- cient to prevent the building up of a negative charge large enough to produce viscosity effects qualitatively similar to those encoun- ease of S III, but that the increase is much smaller, and that 140 Specific Polysaccharides tered in the case of S III (see also Fig. 2); and in SI, also, these forces are only incompletely reduced by isotonic concentrations of salts. The smaller viscosity effects might also be attributed to a lower molecular weight for SI than for S III, but no evidence is available on this point. Although the total viscosity effects of the interionic forces due to the SI anion do not reach the proportions shown by the S III ion, when these forces are depressed by large concentrations of salts or acids, the specific viscosity of S is very close to that of SIII. This is perhaps a reflection of similarities in the arrangement of the sugar and sugar acid units of which the two substances are composed. The low acid equivalent of SI may be taken as evidence that some other explanation must be sought for the widely differing ratios between precipitin index and mouse protection in the case of Type I and Type III antisera than the suggestion of Sobotka and . Friedlander (18) that the ratios are connected with differences in the acid equivalents of the two polysaccharides. Table V shows that in the non-basic specific gum arabic (19) (S.G.A., acid equivalent 835) the accumulation of negative charges is still great enough to give rise to an increase in vai on dilution, while this effect is lost in the case of the specific polysaccharide of Type II pneumococcus (9) (S II, acid equivalent 1250). The specific viscosities of these polysaccharides are also lower than those found at similar concentrations of S I and S ITI, but are higher than that of a non-acidic (and non-basic) bovine tubercle bacillus polysaccharide (B.B.G., Table V). In the series of specific polysaccharides studied, therefore, there appears to be a relation between the magnitude of the viscosity phenomena shown by the substance and the frequency of occur- rence of carboxy! groups in the polysaccharide chain. That the large viscosity effects exhibited by salts of S IIT are due to the con- sequent piling up of negative charges in the salts of this strong multivalent acid is believed to be indicated by the data presented. SUMMARY 1. The specific polysaccharide of Type III pneumococcus is shown to yield a highly ionized sodium salt characterized by a mobile negative ion of very high valence. M. Heidelberger and F. E. Kendall 141 2. Viseosities have been determined for a number of specific polysaccharides under varying conditions of salt concentration, and the findings correlated with the magnitude of the charge on the anion. 8. So strong are the interionic, or Coulomb, forces engendered by the S III polycarboxylate ion that the ratio a increases many times with dilution. At increasing dilutions S ITI solutions show in increasing measure the yield point phenomenon characteristic of plastic solids, an effect.possibly due to the orientation of ions brought about by these forces. 4, The specific polysaccharide of Type I pneumococcus shows viscosity abnormalities of the same character, but of smaller magnitude. Although its acid equivalent is even lower than that of SIII, internal compensation of negative charges by the basic groups present is believed to be the cause of the smaller effect. 5. The data on the specific gum arabic and Type II pneumo- coceus polysaccharide show that the viscosity effects decrease with the relative number of carboxy] groups, or negative charges, in the molecule. BIBLIOGRAPHY . Cf. Heidelberger, M., Physiol. Rev., 7, 107 (1927) for a review. . Heidelberger, M., and Goebel, W. F., J. 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