結合物(PPDs-G)の製法ならびにその性状について (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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Since R. Koch reported tuberculin (OT) in 1891, many attempts have been made to purify tuberculin active principle from culture filtrate of M. tuberculosis or from tubercle bacilli. OT was intended for a therapeutic agent at first, it could not be used clinically because of its adverse effects on tuberculous patients.<BR>One of the writers Ishidate noticed by chance that the aldehyde group of D-glucuronic acid could take the place of that of formaldehyde, and found that the toxicity of various toxins was reduced by incubation with glucuronic acid partially or completely without any significant modification of their antigenicity. Similarly, a preparation of OT (GLT), which was prepared by treating OT with sodium glucuronate (NaG) or glucuronolactone (GL), was reported to affect tuberculous lesions of guinea pigs more slightly by injection than OT.<BR>Considering that active principle of tuberculin exists in its protein component, we prepare glucuronic acid bound PPDs (PPDs-G), using PPDs instead of OT, and compared its physico chemical and biological properties with those of PPDs itself.<BR>PPDs was prepared from culture filtrate of M. tuberculosis Aoyama B strain, which was cultured in Sauton medium for 8 weeks, by 5 to 7 times precipitation with half saturated ammonium sulfate, as described by F. B. Seibert et al. A mixture of PPDs and NaG (containing 3μc of 6<SUP>-14</SUP>CNaG) in the ratio of 1 to 2 by weight and a small amount of antiseptics was incubated at 37°C for 96 to 100 hours. The reaction mixture was then dialysed against distilled water for 30 hours. Dialysate was condensed using Sephadex G 100 and applied to a column of Sephadex G 50. Amount of PPDs in each fraction tube was measured from optical density at 280μE and that of NaG was estimated by 14C radioactivity.<BR>By chromatography of a mixture of PPDs and NaG on Sephadex G 50, both were separated almost completely from each other as seen in Fig. 1.<BR>On the other hand, when PPDs was treated with NaG according to the method described above, elution curve of NaG was observed to be coincided with the second peak (major fraction) of PPDs. This suggests that PPDs combines with NaG. By calculation of binding ratio in each fraction tube, it was found that NaG combined with PPDs at about 1% of the amount of the latter.<BR>PPDs was then lyophilized and subjected to physicochemical analyses.<BR>(a) As seen in Fig.3, PPDs-G moved faster than PPDs in Veronal buffer of μ=0.05, pH8.6 in paper electrophoresis.This suggests that isoelectric point of PPDs-G is lower than that of PPDs.<BR>(b) PPDs-G showed a little smaller value than PPDs in nitrogen content.This suggests that NaG is chemically combined with PPDs.<BR>Biological properties of PPDs-G were as follows:<BR>(a) By tuberculin skin test in sensitized guinea pigs, no significant difference was observed between the potencies of PPDs and PPDs-G.<BR>(b) The infected guinea pigs were injected with either PPDs or PPDs-G.The number of dead animals in the group injected with PPDs-G seemed to be smaller than in the group injected with PPDs.
- 一般社団法人 日本結核病学会の論文
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