The use of performic acid oxidation to facilitate differential staining of epoxy-embedded adenohypophysis

Summary Preoxidation with performic acid facilitated the differential staining of 1–2 thick sections of water-insoluble Durcupan (Swiss Araldite)-embedded adenohypophysis. Alcian blue-PAS-Orange G and aldehyde fuchsin-Orange G staining procedures were used. Except for increases in staining times, these procedures were unmodified from those previously reported for paraffin sections. These techniques permit the light microscopic recognition of two groups of basophils and two groups of acidophils in thick sections cut adjacent to thin sections for electron microscopy.     

Identification of disulfide-containing peptides by performic acid oxidation and mass spectrometry

In addition to reducing the analysis time, the direct examination of proteolytic digests by fast atom bombardment mass spectrometry (FABMS) greatly extends the information that is available from peptide mapping experiments. Mass spectral data are particularly useful for identifying post-translationally modified peptides. For example, the molecular weight of a disulfide-containing peptide may be used to locate the disulfide bond in the protein from which the peptide was derived. This paper describes a new procedure, which is useful for identifying disulfide-bonded peptides. Peptides are treated with performic acid to modify certain residues and thereby cause a characteristic change in the peptide molecular weight. This change in molecular weight is determined by FABMS and used to help identify peptides. Results for a series of small peptides demonstrate that Cys, Met, and Trp are the only residues that undergo a change in molecular weight under the conditions used here. Furthermore, these changes in molecular weight are diagnostic for each of the residues. Cysteinyl-containing peptides are of particular interest, because their identification is essential for locating disulfide bonds. The molecular weight of a peptide increases by 48 mu for each cysteinyl residue present. This approach is used to identify peptides that contain both cysteinyl and cystinyl residues in the peptic digest of bovine insulin. The method is extended to the analysis of a tryptic digest of cyanogen bromide-treated ribonuclease A. A computer-assisted analysis procedure is used to demonstrate the specificity with which peptide molecular weight is related to specific segments of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

The instability of S-beta-(4-pyridylethyl)-L-cysteine to performic acid

S-β-(4-pyridylethyl)-l-cysteine (PEC) was transformed to S-β-(4-pyridylethyl)-l-cysteine sulfone (PEC·SO₂) and to cysteine sulfinic acid following exposure to performic acid. Acid hydrolysis (6n HCl, 110°C, 20 h) of the isolated PEC·SO₂ resulted in its partial decomposition to cysteic acid and alanine. Several minor ninhydrin positive derivatives, including glycine and serine, were also observed.The transformations involving PEC and PEC·SO₂ were followed using high-voltage electrophoresis (HVE) at pH 1.6, 2.0, 3.5, and 6.5 and through use of the amino acid analyzer and gas-liquid chromatography.These studies indicate that as a modification form of the thiol groups of proteins, PEC is not the derivative of choice when subsequent treatment of the protein involves the use of performic acid.     

The amino acid sequence of cysteic acid-containing peptides from performic acid-oxidized ovotransferrin

1. The half-cystine content of ovotransferrin, measured as cysteic acid, was 31mol/80000g of protein. 2. The amino acid sequences of cysteic acid-containing peptides from performic acid-oxidized ovotransferrin were studied. 3. 34 unique cysteic acid residues were identified. 4. It is concluded that hen ovotransferrin does not consist of two identical halves or subunits.     

Improvement of hydrophobic integral membrane protein identification by mild performic acid oxidation-assisted digestion

Integral membrane proteins (IMPs) are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of IMPs make them difficult to analyze. In proteomic analyses, hydrophobic peptides including transmembrane domains are often underrepresented, and this reduces the sequence coverage and reliability of the identified IMPs. Here we report a new strategy, mild performic acid oxidation treatment (mPAOT), for improvement of IMP identification. In the mPAOT strategy, the hydrophobicity of IMPs is significantly decreased by oxidizing their methionine and cysteine residues with performic acid, thereby improving the solubility and enzymolysis of these proteins. The application of the mPAOT strategy to the analysis of IMPs from human nasopharyngeal carcinoma CNE1 cell line demonstrated that many IMPs, including those with high hydrophobicity, could be reliably identified.     

Desaminopenicillamine tocinoic acid derivatives--inhibitors of oxytocin

Tocinoic acid analogs with penicillamine in place of one or both of the cysteine residues have been studied and [1-beta-mercaptopropionic acid, 6-penicillamine] tocinoic acid (dPen6TA) and [1-beta,beta-dimethyl-beta-mercaptopropionic acid, 6-penicillamine] tocinoic acid (dPen1Pen6TA) have been synthesized in solution. Biological activities of these 2 compounds and those of the previously synthesized [1-beta,beta-dimethyl-beta-mercaptopropionic acid] tocinoic acid (dPen1TA) have been assayed. It was found that dPen1TA and dPen1Pen6TA, both of which have a beta,beta-dimethyl-beta-mercaptopropionic acid in position 1, are strong inhibitors of the uterine activity of oxytocin in vitro (without Mg2+) with pA2 values of 7.1 and 7.8, respectively, whereas dPen6TA with penicillamine in position 6 is a mild agonist.