培地組成によるPPDsの物理化学的性状と力価について (精製ツベルクリン(PPDs)とグルクロン酸ナトリウムとの結合-1,2-)

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In part I, the authors reported the preparation of D-glucuronic acid bound PPDs (PPDs-G), and its physicochemical and biological properties. In preparing PPDs-G, we found that PPDs, showed different physicochemical properties among various lots of culture filtrates, and the fact suggests that the combining ratio of PPDs with NaG might vary with PPDs preparations. We therefore examined the culture conditions of tubercle bacilli and the method of preparation of PPDs in order to prepare PPDs-G in homogeneous and reproducible state, and found that modified Sauton medium, which contained twice the amount of asparagine in Sauton medium, was favourable for the preparation of PPDs. This paper presents physicochemical properties and potencies of PPDs obtained from Sauton and modified Sauton media. Fig. 3 shows elution curves of PPDs Lot No.1-No.10 by column chromatography on Sephadex G 50 or G 75. As seen in this figure, PPDs from Sauton showed two different elution. patterns (A or B) according to Lot No, but those from modified Sauton showed similar elution pattern irrespective of Lot No.Physicochemical properties of PPDs preparations before and after chromatography were listed in Table 1. PPDs which showed elution patterns of A or C contained a small amount of polysaccharide or nucleic acid. Therefore slight difference was observed in physicochemical properties before and after chromatography. On the other hand, PPDs which showed elution pattern of B contained about 20% of polysaccharide and 1. 5% of DNA (as 2-desoxy D-ribose), and could be separated into two different fractions, B-I and B-II. The homogeneity of various PPDs preparations described above was examined by discelectrophoresis and ultracentrifugal analysis. An almost single band was observed with each sample in disc electrophoretic pattern, as shown in Fig. 4. Sedimentation analysis using synthetic boundary cell resulted in sharp and symmetrical pattern which suggested the homo geneity of each sample, as seen in Fig. 5. In this connection, molecular weight of each sample was calculated by the method of sedimentation equilibrium and estimated A, B-I, B-II, and C to be 19, 000, 45, 000, 20, 000, 25, 000, respectively. Isoelectric point of each sample was estimated to be 3.7-4.1 by electrofocusing method using carrier Ampholyte, as shown in Fig. 6. According to amino acid analysis, as seen in Table 2, there was slight difference among B-I, B-II and C in amino acid content except glutamic acid and proline. As illustrated in Table I, B-I fraction contained polysaccharide component in 50% of its amount. Using the acid hydrolysate of B-I as test material, we examined the sugar component by paper chromatography, high voltage electrophoresis and gas chromatography, and found that it was mainly composed of D-glucose to D-mannose ratio of about 9: 1. Moreover, we suspected that polysaccharide of B-I would not merely a contaminant but be combined with tuberculoprotein. Furthermore, we compared the tuberculin potencies of various PPDs preparations with that of standard PPDs 15-25 by measurement of the induration of sensitized guinea pigs 24 hours after injection. As shown in Table 3, no significant difference in potency was observed among PPDs preparations. The experimental results thus obtained lead to the conclusion PPDs obtained from modified Sauton medium would be preferable to that from Sauton medium, since the tuberculin potencies of the former PPDs are invariable from Lot to Lot.
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培地組成によるPPDsの物理化学的性状と力価について (精製ツベルクリン(PPDs)とグルクロン酸ナトリウムとの結合-1,2-)

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