RESYNTHESE UND WIEDERGEWINNUNG VON KRISTALLINEM INSULIN AUS DEM REl~OMBINATIONSANSATZ NATORLICMER .A-UND B-KETTE J. JI~N'WX-I * Imtitut ffir Pi~.vsiologiscl~c Clredc, UnivevsitUt Ttibin~en, 74 TUingcn (B,Z?.D.) (Eingcgnngcn am 27. Soptombcr 1872) SUM?+lARY
Resynthesis nm? recovery of crystalZinc insulin from the recombination m&We of natural A and B drains
The quantitative recovery of biologically active crystalline bovine insulin from recombination products of natural A and 13 chains is described.
S-Sulfonated A chain from bovine insulin was reduced with mercaptoethanol to the corresponding sulfhydryl form followed by recombination with natural S-sulfonatccl B chain in a 2: I weight ratio and neutralization of the recombination mixture to pH 7.0, The insulin activity was found to be 34 o/o in the recombination misture, based on the amount of I3 chain used, as shown in the mouse convulsion test. The solution was immediately lyophilized to a small volume and acidified, The precipitate and supcrnatant were chromatographed by gel filtration on Sephadex G-go with I N acetic acid and the original amount of insulin (34 %) was recovered. The activities of the two insulin portions were: after purification of the supernatant 100 %, after purification of the precipitate 90 %. The insulin isolated by this method is identical with the natural hormone with respect to gel chromatographic mobility, ccllogel electrophoretic mobility, biological activity, crystalline form and amino acid composition, The method is simple and reproducible.
It was not possible to separate a hybrid insulin from the heterogeneous protein mixture synthesized by the Merrifield method, The possible causes are discussed. BINLEITUNC Seit der Synthese des Insulins14 im Jahre 1963 arbeitet man intensiv an der Verbesserung der lnsulinausbcute bei der A-und B-Kettcn-Rekombination'-0. Die Vercinigung reduziertcr nattirlicher Ketten durch oxydative Regenerierung flihrt nur ,.in sehr geringem Masse zu aktivcm Insulin; haupt&tchlich entstehen dabei Kettenpolymerisationsprodukte und Insulin-I&mere, DIXON UND WARDLAW'~ , sowie Du und Mitarb." liberflihrten die vollsttlndig getrennten Ketten in die S-Sulfonate, nach Reinigung in die SW-Ketten und vereinigten diese oxydativ bei pH 9 zu einem Rekombinat mit I-Z o/0 Insulingehalt. Den chinesischen Autoren gelang l Gcgcrlwlirtigc