Further Studies on the Heterogenetic Haptene.

Abstract

In continuation of the studies recorded previously 1 the following observations were made.

The crude material obtained from horse kidney by extraction with alcohol, and freed from ether soluble substances in the manner described previously, was extracted with boiling benzene. From the solution on standing a precipitate separated out which was removed by centrifugalization. The solution was concentrated. On addition of an equal volume of alcohol a precipitate formed. This was dissolved in benzene and precipitated with alcohol, and the procedure was repeated many times until there appeared in the benzene solution a substance giving a distinct copper orcinol reaction. The final precipitate was dissolved in water and precipitated from the solution with two volumes alcohol, and was then extracted thoroughly with methyl alcohol and with chloroform. The fraction soluble in methyl alcohol (A) was water soluble and gave a strong orcinol copper test. The insoluble part (B) did not give this reaction and was practically insoluble in organic solvents. With hot water it yielded a viscous pseudosolution. This solution gave a flocoulent precipitate on addition of Fehling's reagent. On hydrolysis of “B” with 3 per cent H₂SO₄, after about 30 minutes, an insoluble substance settled out and the solution reduced Fehling's reagent and gave a crystallized osazone. After hydrolysis for 12 hours the quantity of sugar amounted to about 35 per cent, calculated as glucose. After precipitation with Fehling's solution substance B contained neither P nor S. The material was active in the complement fixation test with heterogenetic immune serum and produced heterogenetic hemolysins in rabbits when several injections of 1 mg. each of the substance mixed with pig serum were given. Even quantities as small as 0.2 mg. had still noticeably immunizing properties.