Christian Birr

Antibodies against synthetic peptides as a tool for functional analysis of the transforming protein pp60src

To study the function of pp60src, the transforming protein encoded for by Rous sarcoma virus, we have raised antibodies against synthetic oligopeptides corresponding to the primary structure of pp60src. All eight investigated peptides were immunogenic in rabbits, and four induced pp60src-specific antibodies. We screened tumor-bearing rabbit (TBR) sera for antibodies against the peptides; this revealed that five out of six of the peptides, chosen according to a high hydrophilicity plot, were related to epitopes of native pp60src, in contrast to two peptides of low hydrophilicity, which contained a cleavage site for protease. Antibodies against three of the peptides appeared to react with the kinase-active site of pp60src, as these antibodies were phosphorylated in their heavy chain upon immune precipitation. Antibodies against two of the peptides, in contrast to the others, did not precipitate pp60src when this molecule was complexed with two cellular proteins, pp50 and pp90. This observation allows speculation about the location of the pp60src site involved in the formation of this complex.

Direct conversion to pure PTH-amino acids without manipulations in the solid phase edman degradation (Laursen Variant) of peptides

The Edman degradation of peptides and proteins [2] in solution and in heterogeneous phase can be precisely performed automatically at the present time [3]. Usually the automatic process on each stage of degradation ends with the cleavage and extraction of an amino acid thiazolinone, which subsequently has to be manually converted into the characteristic phenylthiohydantione of the amino acid (PTH) before identification. The quantity and purity of each PTH derivative, which determine the accuracy of PTH identification and therefore the reliability of sequence analysis, both depend upon completion of all particular chemical reactions during degradation and conversion and of their entire protection from oxygen. Therefore each saved manipulation leads to a methodical advantage. Except for direct conversion of thiazolinones to PTH-amino acids the Laursen method [4] of Edman degradation on peptides bound to a solid phase satisfies best the demands for precise sequence determination. We succeeded in eliminating these remaining shortcomings of manual conversion and purification: Pure PTH-amino acids are directly obtained from automatic degradation of peptides on solid phase by injecting H20 to the solution of the thiazolinones in TFA and passing the mixture through an additional heated reaction coil for conversion. The quantitative identification of the PTHs is performed by high pressure liquid chromatography (HPLC), which in a single run permits the separation

4Phe—6Val—antamanide, an antitoxic cyclodecapeptide with C2 symmetry☆

The cyclopeptide II, in contrast to I, has C2 symmetry and was expected to lend itself to a less difficult structure analysis than I. However, cyclopeptide II is of interest only if it also has antitoxic activity against phallotoxins. It is also possible to obtain an achiral cyclodecapeptide by substitution in I of lVa1 by Ala and an exchange of amino acid 4 + 6. However, the ‘Ala variant of I previously proved less than 5% as active as I, whereas substitution of 4Ala by Val resulted in practically no loss of activity [3] .

Peptidsynthesen, LVI. Verwendung symmetrischer Boc-aminosäureanhydride

Symmetrische Boc-aminosaureanhydride werden aus den Natriumsalzen der Boc-aminosauren in Tetrahydrofuran oder aus den Triathylammoniumsalzen in Methylendichlorid mit Phosgen hergestellt und in situ mit verschiedenen Aminosaure- oder Peptid-methylestern zu Boc-peptidestern gekuppelt. Die Ausbeuten sind mit den mit gemischten Anhydriden erhaltenen vergleichbar (siehe Tabelle 1). Peptide Syntheses, LVI. — Application of Symmetric Anhydrides of Boc-amino Acids Symmetrical anhydrides of Boc-amino acids are prepared by reaction of sodium salts of Boc-amino acids in tetrahydrofuran or triethylammonium salts in methylene dichloride with phosgene. They are coupled in situ with several amino acid or peptide methyl esters to yield Boc-peptide esters in amounts comparable with those obtained by the mixed anhydride method (see Table 1).

Automatization of the Merrifield Peptide Synthesis

Already in the incipience period of Merrifield’s method it was demonstrated that the new idea included the possibility for automatization of most of the chemical operations necessary for peptide synthesis [39]. On this aspect was based a great part of the enthusiasm which still accompanies the development of the methodology to date: Like a fata morgana all routine work in conventional peptide synthesis seemed to be transferable to a more or less automatically operating machine. The only remaining labor was to refill reagents and solvents, to maintain the instrument, and to reap the end product.

Chemical Details of the Method

The selection of methodical procedures noticed in this chapter should not constitute a devaluation of the scientific-intellectual wealth of the approaches and modifications omitted here, which often are chemically fascinating but less generally applicable from the point of view of practical preparative use. For the same reason, several details of the original method will be mentioned only in passing, since some of them became obsolete by later discovered aspects.

Critical View on the Applicability of the Merrifield Synthesis

In this final chapter it is not the author’s intention to add to the existing excellent and very comprehensive reviews of [33, 35] applications of gel phase peptide synthesis another less complete one. Instead, from a very personal view, some points are selected for discussion on the applicability of the Merrifield principle, which is of indubitable wealth, not at all only in peptide chemistry.