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Vol. 45 POLYSACCHARIDES FROM PENICILLIUM LUTEUM SERIES 189 Morgan, W. T. J. (1938). Helv. chim. Acta, 21, 469. Shaffer, P. A. & Hartmann, A. F. (1921). J. biol. Chem. Neuberg, C. (1907). Biochem. Z. 8, 519. 45, 365. Ogston, A. C. (1949). Biochem. J. 45, 189. Somogyi, M. (1937). J. biol. Chem. 117, 771. Partridge, S. M. (1946). Nature, Lond., 158, 270. Stacey, M. (1947). J. chem. Soc. p. 853. Partridge, S. M. (1948). Biochem. J. 42, 238, 251. Thom, C. (1930). The PeniciUia. London: Bailli6re, Tindall Raistrick, H. & Rintoul, M. L. (1931). Philos. Tranm. B, and Cox. 220, 255. Wise, L. E. & Appling, J. W. (1944). Indu8tr. Engng ehem. Reclaire, A. (1908). Ber. dt8ch. chem. Ge.s 41, 3665. (Anal. ed.), 16, 28. The Sedimentation and Diffusion of Polysaccharides from Penicillium luteum; Interpretation of the Results Obtained from Polydisperse Material in the Gouy Diffusiometer BY A. G. OGSTON Department of Biochemi8try, Univer8ity of Oxford (Received 18 February 1949) Samples of the dextro- and laevo-rotatory poly- Both samples proved to be polydisperse. The dextro- saccharides, prepared from Penicillium luteum by rotatory material (Fig. la) was composed of two main Freeman & Macpherson (1949), were submitted for fractions: a more slowly sedimenting fraction which appeared the examination of their ultracentrifugal sedimenta- to be homogeneous and a more rapidly sedimenting fraction which was heterogeneous. The sedimentation constant of the tion and diffusion. faster material and the relative proportions of the two fractions could not be determined accurately because the EXPERIMENTAL boundaries were not clearly resolved. However, separation of the schlieren curve into two more or less symmetrical The solid materials were dissolved in, and thoroughly parts (Fig. 2) gave an approximate estimate of the amounts dialysed against, buffer of composition: NaCl, 02M; of the two components. The combined boundary repre- KH2PO4,0*027 M; Na2HPO4, 0*027M. The final concentrations sented only 0-86 of the total refracting material. were about 1 g./100 ml. and were estimated by refractometry The laevorotatory material (Fig. 1 b) gave a single boundary, against the buffer. the thickness of which showed that the polysaccharide was Meniscus polydisperse; it consisted of material sedimenting over a range of rates, symmetrically distributed about a mean value. Integration of this boundary showed that it repre- / Meniscus dc. (a) 4% zk X tdx Base line - x Fig. 2. Tracing from sedimentation diagram (full line) of dextrorotatory polysaccharide, 47 min. after reaching full speed (60,000 rev./min.), to show analysisinto components (broken lines); gradient of concentration dcld/ against (b) position in cell, x. Fig. 1. Sedimentation diagrams: (a) dextrorotatory poly- sented only 0 64 of the total refracting material; however, saccharide 37 min. afterreaching full speed; (b) laevo- this value may be too low, because the lower limit of the rotatory polysaccharide 80 min. after reaching full speed boundary appeared to be reaching the bottom of the cell (60,000 rev./min.). before its upper limit had fully left the meniscus. The results are given in Table 1. Sedimentation measuremente. Sedimentation was observed Diffusion measurements. Diffusion was measured by means in a Svedberg oil-turbine ultracentrifuge by the method of of the Gouy diffusiometer (Coulson, Cox, Ogston & Philpot, Philpot (1938), using the standard conditions of running 1948). Ogston (1949) has shown that this method can be recommended by Cecil & Ogston (1948). used to determine the diffusion constants of a mixture of two 190 A. G. OGSTON I949 Table 1. Data obtained from utltracentrifugal 8edimeentation Fraction of total refracting material in Proportion of ultracentrifuge component in Material Component diagram diagram S20(co,). X 1013 Dextrorotatory (1) Monodisperse 0-86 043 4-13 (2) Polydisperse 057 5-2 (approx.) Laevorotatory Polydisperse 0*64 1*79 Table 2. Data obtained from diffu8ion Proportion Material Component of component D20(or.) x 107 Approx. mol. wt. Dextrorotatory (1) Monodisperse 0-27 5.5 50,000 (2) Polydisperse 0-73 1-9 170,000 Laveorotatory Polydisperse Av. 8*32 15,000 homogeneous components. Provided that one of the com- boundary at distances + x and x from its centre and the ponents is present only in small amount, the error introduced angular deflexion 0, of such light at time t are given by by its polydispersity into the estimate of the diffusion constant of a homogeneous main component is small; 2v r =- S {cz f (z.)}p however, if, as in the case of the dextrorotatory poly- saccharide, a main component is polydisperse, considerable where errors are introduced into the estimates of the diffusion f (zi) = e" dz -zie-zi' and z;=x/V(4D,t); constants of both components and of their proportions. The o values quoted in Table 2 are, therefore, only rough and it is not surprising that the proportion of the components and Or AS{V(47rDit) = ) estimated from the diffusion data differs from that obtained from the sedimentation diagram (Table 1). It follows that -2x/A. Any attempt to analyse the diffusion data given by a highly polydisperse material, such as the laevorotatory poly- saccharide, in terms of two homogenpous components, is of Now Dt =S (o4Di) t jx = little value and could do no more than indicate the range of polydispersity. It would be more useful to estimate the mean diffusion constant, for comparison with the mean sedi- =| Acx ar a2 dx ae c fr dx mentation constant. However, the mean diffusion constant which is obtained from the movement of the outermost (1) - =24 | tR. {ao interference band by the Gouy method is not the weighted mean diffusion constant, but is given by This quantity can be approximately computed from the Gouy interference pattern by the following procedure. The 1 ai values of r for the interference minima, from without inwards are i, 1 + .; the corresponding displacements of , .. where ;i is the fraction of material having diffusion constant the minima from the optic axis, X, are measured at a given Di. Use of this value, together with the mean sedimentation time t. In addition, the maximal displacement XZ,,. is constant, to calculate the molecular weight would yield a calculated from Xo/l,o (Coulson et al. 1948; Ogston, 1949), rather curious mean value. A method has therefore been which corresponds to r=0. r, Xrso 8r., 8xr 0 (8) and found, and is described below, for obtaining the arithmetic- mean diffusion constant from the Gouy data. The result of a are then tabulated. Since =X/F, where F is the this calculation is given in Table 2. focal distance, equation (1) is approximated by F2A2 THEORETICAL Dt= S X18- 24v S 58X Method of obtaining an arithmetic-mean diffu8ion This sum is computed over the whole range of interference constant by the Gouy method bands, including the optic axis where X =0 and r =v, but Where the diffusion boundary is made up of a range of omitting the interference minimum next to the optic axis, superimposed Gaussian boundaries, if;i is the fraction of the since its proper value of r is uncertain (Kegeles & Gosting, total refractive increment due to each component i having 1947). By thus computing Dt at two or more values of t, diffusion constant Di and if v is the total refractive increment the value of D1 is obtained. (expressed as numbers of wavelengths of phase difference), This method was tested on a record obtained with a nearly then the phas difference r of light passing through the homogeneous sample of lactoglobulin. The mean diffusion Vol. 45 SEDIMENTATION AND DIFFUSION OF POLYSACCHARIDES 191 constant (in buffer at 200) obtained from 1s -= was given in Table 2. These were derived from the sedimentation and diffusion constants assuming a 7415 x 10-7, while the application of the above method gave value of 0-62 for the partial specific volumes. D =694 and 7-02 x 10-7, from two intervals of time. The The value of the mean diffusion constant of the mean diffusion constant of a mixed solution of lactoglobulin laevorotatory polysaccharide, calculated by the and sucrose (Ogston, 1949) was found to be 10-82 x 10-7, the method described, would give a reliable estimate of expected value being 10-85 x 10-7. its mean molecular weight if it were certain that the average applied to the same range of material as does DISCUSSION the estimate of the sedimentation constant. The fact It is clear from the sedimentation diagrams that both that only 064 of the refracting material appears in polysaccharides are polydisperse, the dextrorota- the sedimentation boundary shows that this may not tory consisting of homogeneous and heterogeneous be so. fractions and the laevorotatory being entirely SUMMARY heterogeneous. In addition, in neither case does all the refracting material appear in the sedimentation 1. Measurements of the sedimentation and boundaries, which indicates that a proportion of the diffusion of two samples of polysaccharide from material may sediment too quickly or too slowly or Penicillium luteum are described. may be too highly polydisperse to contribute to the 2. A new method is given for analysing the boundary diagrams. Thus, while the mean sedi- diffusion data obtained with the Gouy diffusio- mentation constants of the material can be regarded meter, so as to obtain a value of the arithmetic-mean as established, in view of the uncertainties and errors diffusion constant which is comparable with the discussed above neither the proportions of the mean sedimentation constant. components nor their diffusion constants should be 3. Approximate values for the amounts and regarded as more than rough estimates, and the constants of the components of the polysaccharides same is true of the estimates of molecular weights, have been deduced. REFERENCES Cecil, R. & Ogston, A. G. (1948). Biochem. J. 43, 592. Kegeles, G. & Gosting, L. J. (1947). J. Amer. chem. Soc. 69, Coulson, C. A., Cox, J. T., Ogston, A. G. & Philpot, J. St L. 2516. (1948). Proc. Roy. Soc. A, 192, 382. Ogston, A. G. (1949). Proc. Roy. Soc. A, 196, 272. Freeman, G. G. & Macpherson, C. S. (1949). Biochem. J. Philpot, J. St L. (1938). Nature, Lond., 141, 283. 45, 179. Metabolic Products of Trichothecium roseum Link BY G. G. FREEMAN AND R. I. MORRISON, Imperial Chemical Indu8trie8 Limited, Nobel Divi8ion, Research Department, Stevenston, Ayrshire AND S. E. MICHAEL, Department of Biochemi8try, London School of Hygiene and Tropical Medicine, London, W.C. 1 (Received 22 February 1949) The work described in this paper was carried out rosein I and rosein II, while the name rosein III is independently at Stevenston and the London School proposed for the third compound which has been of Hygiene and Tropical Medicine. When the found only in the culture fluid. The three new pro- separate investigations were communicated to the ducts are additional to trichothecin, the antifungal Biochemical Society (Freeman & Morrison, 1948; substance which was described by Freeman & Michael, 1948) the results from the two laboratories Morrison (1949a). were found to be so similar that it was decided to It has been known for many years that fruits present a joint account of the work. This paper deals attacked by the pink rot caused by T. roseum Link with the isolation of three crystalline metabolic contain a bitter principle. The latter was isolated in products of Trichothecium ro8eum which had not the form of a crude syrup and its solubility described hitherto been described. Two of these products by Iwanoff (1904). Antagonism between T. ro8eum which are present mainly in the mycelium, and only and certain plant pathogenic fungi was reported by in smaller amounts in the culture fluid, were named Whetzel (1909), Boning (1933), Koch (1934) and

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