Joachim Jentsch

The use of a new silica gel separation system in thin-layer chromatography.

SummaryA new method for the chromatography of amino acids is described in which D- or L-amino acids are separated on ICT-Empore thin-layers. The compounds are developed ascending by means of normally used solvent systems. An overloading of the plates is nearly impossible. On the other hand, hydrophilic amino acids are well separated. A second front, moving with these amino acids and emerging with ninhydrin stain, was not detectable.

Weitere Untersuchungen zur Aminosäuresequenz des Melittins

Melittin is the (main) toxic peptide of bee venom having a molecular weight of 2840, with a known sequence (s. fig. 1). Optical rotatory dispersion of non-crystalline melittin in aqueous solution suggests that the polypeptide chain is random, although 7% α-helix has been determined. These results are in agreement with the amino acid sequence of melittin and the assumption that the biological activity is attributable to its surface active character.

Resynthese und wiedergewinnung von kristallinem insulin aus dem rekombinationsansatz natürlicher A- und B-kette

Abstract Resynthesis and recovery of crystalline insulin from the recombination mixture of natural A and B chains The quantitative recovery of biologically active crystalline bovine insulin from recombination products of natural A and B 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-sulfonated B chain in a 2:1 weight ratio and neutralization of the recombination mixture to pH 7.0. The insulin activity was found to be 34 % in the recombination mixture, based on the amount of B chain used, as shown in the mouse convulsion test. The solution was immediately lyophilized to a small volume and acidified. The precipitate and supernatant were chromatographed by gel filtration on Sephadex G-50 with 1 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, cellogel 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.

Enzymatische Spaltung nativer cystinhaltiger Polypeptide durch Thermolysin (E.S. 3.4.4.) / Enzymatic Hydrolysis of Native Cystine-containing Polypeptides with Thermolysin

A new method is described for separating cystine peptides from other peptides after hydrolysis of native bovine insulin by the specific protease thermolysin using paper shields instead of column chromatography. The application of this method to sequence determination of other species insulins is possible. In contrast to the proteolytic fraction (α-protease) from Crotalus atrox venom, thermolysin rapidly hydrolyzes all susceptible peptide bonds in native insulin in high yields. Therefore, the amounts of substrate required to sequence determination are low compared with the known methods involving the denaturation and chemical splitting of polypeptides before proteolysis, such as the mercaptolysis of insulin, carboxymethylation of the A- and B-chain and column chromatography of the reaction products. The yields of the isolated chains are too low for further enzymatic digestion and analysis if the substrate is available only in minimal quantities. Instead of thermolysin, it is in principle possible to use any other bacterial protease, e. g. subtilisin, but the products of hydrolysis of insulin are easily soluble in water so that the fractionation in soluble and insoluble peptides before isolation is not possible. Thus, thermolysin is a valuable tool in sequence analysis of cystine-containing polypeptides.