Diffusion of synthetic and natural polyphosphates

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

76, 396-402 (1958)

Ditrusion of Synthetic and Natural Polyphosphates’ B. J. Katchman2 and H. E. Smith3 From the Mound

Laboratory,

Miamisburg,

Ohio

Received August 26, 1957

INTRODUCTION

The existence of naturally occurring polyphosphates in certain bacteria, plants, and lower animals has been known for many years (1). It has been shown that there are at least two polyphosphate fractions in yeast which appear to have differing metabolic activities (2-5). Recently, it has been shown that the so-called “insoluble” polyphosphate fraction of Chlorella is involved in photosynthesis (6). Furthermore, work in this laboratory indicates that the “insoluble” polyphosphate fraction of Saccharmyces cerevisiaeis involved in cellular division> It has been suggested (7) that these two polyphosphate fractions in yeast might differ in chain length. This would explain the differential separation, in a trichloroacetic acid-protein medium, of these polyphosphates into “insoluble” and “soluble” fractions. The polyphosphate fractions in yeast have been identified with the synthetic inorganic polyphosphates because both are made up of labile phosphorus linkages which hydrolyze in 1 N acid at 100°C. in 10 min., are precipitated by barium ion in acid media, and give the characteristic metachromatic reaction. While the chain length of the “soluble” polyphosphate fraction has been determined by end-group titration, no data have been obtained for the chain length of the “insoluble” polyphosphate fraction, nor is there any information available as to the physicochemical identity of the natural and synthetic polyphosphates. * Abstracted from MLM-1052, June 10, 1955, and given at 130th Meeting of American Chemical Society, September, 1956. Contribution from Mound Laboratory, Miamisburg, Ohio, which is operated for the U. S. Atomic Energy Commission by the Monsanto Chemical Company under Contract No. AT-33-l-GEN-

53. * Present address: Department of Research, Miami Valley Hospital, Ohio. 8 Present address: Kurth Malting Company, Milwaukee, Wisconsin. 4 Katchman, B. J., and Fetty, W. O., in preparation. 396

Dayton,

DIFFUSION OF POLYPHOSPHATES

397

This paper reports the results obtained from a study of the diffusivity of synthetic and naturally occurring polyphosphates. There is every indication that the natural and synthetic polyphosphates behave similarly and that the diffusivity and chain length can be correlated under certain experimental conditions. There is evidence that the “insoluble” polyphosphate fraction from yeast has a considerably longer chain length than the “soluble” fraction. EXPERIMENTAL The diffusion studies were made using Perkin-Elmer Tiselius apparatus with a schlieren optical system and a 2-ml. analytical cell. The experiments with sodium polyphosphates were performed at 22 f 1°C.; the free-acid form experiments were performed at 4°C. Except as noted, all experiments were performed by dissolving the samples in distilled water and diffusing against distilled water. The synthetic poly- and metaphosphates were obtained from Dr. John It. Van Wazer, Monsanto Chemical Company. The yeast polyphosphates were isolated by trichloroacetic acid extraction of cultures of Saccharomyces cerevisiae. The details of the method of extraction and the conditions for growth and harvesting of the yeast cultures are reported elsewhere (5). The unique conditions of growth which are necessary before appreciable amounts of the “insoluble” polyphosphate can be obtained limited the amount available for study. The free-acid forms were obtained by cation exchange of the sodium or barium salts with a charged cationic resin of the type Amberlite IR 112. All operations were performed at 4°C. to minimize hydrolysis and chain scission. The polyphosphates were free of 260.rnr absorbing material and organic carbon contamination. All other salts investigated were commercially available analytical reagent grade. The experiments with the potassium Kurrol salts were performed after 24 hr. dialysis against the solvent, which was either water or 0.1 M sodium chloride. The initial boundary which was formed was compensated into the center of the cell by means of an automatic compensator with a syringe attachment at the rate of 3 cm./hr. Although these diffusion boundaries were imperfect, no attempt was made to sharpen them. Except as noted, the refractive-index gradients were Gaussian in form; many curves were checked by mathematical analysis (8). Diffusion coefficients may be calculated when the initial boundary is diffuse, providing it is Gaussian, by using the method of differences; this is satisfactory when spreading is large (9). Diffusion coefficients were calculated by the method of differences with an average variation of less than 3% in any one experiment. The diffusion coefficients reported here are the averages of several separate experiments; the average value is within 5% of the value obtained in each individual experiment. Photographs of the diffusion cell were taken on Ansco isopan film and were enlarged and traced. The diffusion coefficients were calculated from the enlarged tracings from the area and maximum height by means of a standard equation (8).

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Chain-length determinations were made by means of end-group titrations and were designated as it (10). The concentrations of the phosphate solutions are reported as grams of phosphorus/100 ml. of solution. Orthophosphate was determined by a calorimetric method (11). The condensed phosphates were hydrolyzed to orthophosphate by boiling aliquots of the phosphate solutions in 1 N hydrochloric acid for 10 min. on a water bath. In the case of the free-acid forms of the polyphosphates, diffusion measurements were made at 4°C.; however, for comparison purposes the diffusion coefficients have been corrected to 22°C. with the aid of a standard equation (12). RESULTS

The diffusion coefficients, D, of the phosphate salts are tabulated in Tables I and II. The data obtained for the free-acid forms of the synthetic polyphosphates fi 16, A 36, r~85, and ri. 105, and of the “soluble” and “insoluble” yeast polyphosphates are plotted in Fig. 1 as D vs. concentration in grams phosphorus/100 ml. With the exception of the potassium Kurrol salts, all phosphates gave refractive-index gradients which were Gaussian in form. The Kurrol salts exhibited skewed refractive-index curves even in the presence of electrolyte. Only the Kurrol salts showed asymmetry in the refractive index curves similar to that reported for elongated, linear polymers (13). In general, the diffusion coefficients decreased as the molecular size increased, as, for example, the series of sodium phosphates in Table I from pyro- up to the polyphosphate ?i 36, and the series mono-, di-, and tripotassium phosphate in Table II. However, no distinction could be TABLE Diffusion

Coeficients

of Meta-

I and Polyphosphates Concentration g. P/100 ml.

Sodium trimetaphosphate Sodium tetrametaphosphate Tetrasodium pyrophosphate Sodium tripolyphosphate Graham’s salts A 16 ii 36 A 85 A 105 Kurrol salts” ri. 1600 n 1600

0.581 0.380 0.400 0.400 1.06 0.400 0.400 0.400 0.200 0.200

at RF’C. D x 106 sq. cm.lsec.

7.52 7.79 7.56 7.09 5.54 5.16 5.25 5.16 5.06 0.351b

QGradient curves of Kurrol salts were skewed. 6 Twenty-four-hr. dialysis and diffusion against 0.1 M sodium chloride.

DIFFUSION

OF

TABLE Diffusion

Coeficients

399

POLYPHOSPHATES

II

and Sodium Phosphates

of Potassium

at 22°C. D X 106 sq. cm./rec.

Concentration

g. P/IO0 ml

0.400-0.775 0.388-0.775 0.400 0.334-0.400

Potassium dihydrogen phosphate Potassium monohydrogen phosphate Potassium orthophosphate Sodium orthophosphate decahydrate

9.96 9.54 9.25 6.61

made between 6- and S-membered rings since the trimet’a- and tetrametaphosphates had diffusion coefficients that were nearly identical as shown in Table I. Tripolyphosphate diffused less rapidly than did trimetaphosphate, although the reverse might be expected from structural considerations alone. The most striking of the results shown in Table I is the plateau formed in the diffusion coefficients of the polyphosphates above ri. 36. Thus, above a chain length fi 36, the apparent diffusion coefficient is indcpendent of chain length. The effect of the counter ion on the diffusion coefficient is quite marked

I2 -

6It

0

II

.I

.2

I1

.3

A

.5

CONCENTRATION

I

I

I

I

I

I

.6

.7

.6

.9

I.0

I.1

(GMS

I

1.2

I

1.3

P/ 100 ML)

FIG. 1. Diffusion coefficients vs. concentration of the free-acid forms of polyphosphates at 22°C. The curves through the dots are plots of D vs. concentration for the synthetic polyphosphates fi 16, fi 36, and ri. 85. The open circles represent. data for the acidsoluble yeast fraction; the closed circle, the acid-insoluble yeast fraction; and the triangle, synthetic polgphosphate fi 105. All the data plotted were obtained at 4°C. and have been corrected to 22°C.

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SMITH

in the case of the potassium and sodium orthophosphates. These coefficients are 9.25 X 10e6and 6.61 X 1OP sq. cm./sec., respectively. The effect of electrolyte upon the diffusion coefficient was demonstrated by the Kurrol salts. Diffusion of fi 1600 in water against water is more than ten times more rapid than diffusion in salt solution against salt solution. Concentrations of salt large enough to provide gradient curves that were not skewed were not determined. The diffusion coefficients for the sodium polyphosphates at constant concentration show no variation with chain length above A 36. To study the effect of concentration of the diffusivity of the polyphosphates, the free-acid forms were investigated; the yeast polyphosphates are more readily obtained in this form than as sodium salts in the fractionation procedure. The diffusion coefficients, corrected to 22”C., are shown plotted against concentration in Fig. 1. In dilute solution, D vs. concentration is linear, and the slopes of fi 16, ?i 36, and ?i 85 can be readily distinguished. Accurate measurements could not be obtained at lower concentrations with the equipment used in these experiments. At higher concentrations the curves merge and plateau. Although not represented in Fig. 1, it was found that in concentrated solutions containing about 1.3 g. phosphorus/100 ml., D begins to decrease. The values obtained for the “soluble” and “insoluble” yeast polyphosphates are plotted in Fig. 1. The soluble fraction falls in the A 16 class and is in good agreement with titration data (6 = 20 f 2). The “insoluble” fraction falls in the class fi 85. This is in agreement with titration data.6 In three experiments, the calculated fi was 60 f 20. DISCUSSION

The decrease in the diffusion coefficients with increased chain length of the series of sodium phosphates (pyro- through ii 105) is not surprising; however, the plateauing of the diffusion coefficients above ?i 36 is unique. Wall and Doremus (14) have shown that the fraction of cations bound to polyions increases with polymer chain length, thus: tetrasodium pyrophosphate, 15-20% bound; and, ?i 150, 70 % bound. Thus, a limiting degree of cation association above fi 36 may be a factor in the apparent plateauing of diffusion. An increased rate of diffusion, as concentration is decreased,is demonstrated for inorganic polyphosphates. This type of behavior is known to 6Katchman, B. J., and Talley, L. H., unpublished data.

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401

occur in other polymer systems. It has been suggested that mutual entanglement of elongated particles would retard motion and that dilution would cause disentanglement with a resulting increase in the rate of diffusion (8). This explanation is also consonant with the plateauing, at higher concentrations, of the diffusion vs. concentration curves. The D vs. concentration curves for the longer chains intersect or merge with the plateau at lower concentrations than do the shorter chains. This suggests the concept of a limiting degree of entanglement. In this connection one might consider the effect which dilution has, in a general sense, upon the degree of association of cations and its dependence upon chain length. These two factors, degree of entanglement and degree of association, may be interdependent. The data presented here indicate the likelihood that the two polyphosphate fractions in yeast actually differ in chain length. It is suggested that specificity arising from the effect which chain length may have on the physicochemical properties of the polyphosphates may explain the differing biological activity exhibited by the “soluble” and “insoluble” polyphosphates in yeast. The apparent specificity, in a met,abolic sense, which the longer chain polyphosphate has may be explained in part by the effect of chain length upon protein-polyphosphate interactions. However, in view of the high degree of cation association which long-chain polyphosphates exhibit, immobilization of large amounts of intracellular cations may be effected by the buildup of intracellular long-chain polyphosphates. These two factors may function in cellular control of yeast growth. SUMMARY

The diffusion coefficients (D) of synthetic and yeast polyphosphates have been determined. The “soluble” yeast fraction can be identified with a chain length of about 16 phosphate units and the “insoluble” yeast fract,ion with a chain length considerably higher, probably around 85 units. In dilute solutions of the free acid forms of the polyphosphates, the variation of D with concentration is linear over the range 0.1-0.4 g. phosphorus/100 ml. In this range the slope of D vs. concentration for each of the classes of polyphosphates studied was unique enough to permit differentiation between the classes of polyphosphates. It is suggested that the diffusion of polyphosphates is influenced by cation association.

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KATCHMAN AND SMITH REFERENCES

1. SCHMIDT, G., in “Phosphorus Metabolism” (W. D. McElroy and B. Glass, eds.). Johns Hopkins Press, Baltimore, 1951. 2. MANN, T., Biochem. J. 38, 339, 345 (1945). 3. JUNI, E., KAMEN, M.D., SPIEGELMAN, S., AND WIAME, J., Nature 180, 717 (1947). 4. WIAME, J. M., J. Biol. Chem. 178,919 (1949). 5. KATCHMAN, B. J., AND FETTY, W. O., J. Bacterial. 69, 607 (1955). 6. WINTERMANS, J. F. G. M., Mededel. Landbouwhogeschool Wageningen 66, 2, 69 (1955).

7. KATCHMAN, B. J., AND VAN WAZER, J. R., Biochim. et Biophys. Acta 14, 445 (1954). 8. GEDDES, A. L., in “Physical Methods of Organic Chemistry” (R. Weissberger, ed.), Vol. 1, p. 301. Interscience Publ. New York, 1945. 9. ALBERTY, R. A., J. Am. Chem. Sot. 70, 1675 (1948). 10. VAN WAZER, J. R., KAMEN, M. D., SPIEGELMAN, S., AND WIAME, J., J. Am. Chem. Sot. 72, 639, 644,647, 655, 906 (1950). 11. FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 66,375 (1925). 12. COHN, E. J., AND EDSALL, J. J., “Proteins, Amino Acids, and Peptides,” p. 410. Reinhold Publ. Corp., New York, 1943. 13. NEURATH, H., AND SAUM, A. M., J. Biol. Chem. 126,438 (1938). 14. WALL, F. J., AND DOREMUS, R. H., J. Am. Chem. Sot. 76, 868 (1954).