Francesco Chillemi

Isoelectric focusing of peptides

Abstract Analytical isoelectric focusing of peptides in 0.7-mm thick gel slabs, in presence of 8 M urea, is made possible by staining the gel directly in Coomassie Brilliant Blue G-250 dissolved in 1 N H2SO4—12% trichloroacetic acid solution. The carrier ampholytes are soluble in this solvent whereas the peptides form a macromolecular aggregate with the dye, thus being trapped and precipitated within the gel matrix. The minimum critical peptided length for precipitation and staining is of the order of 15 amino acids. Below this length, only some basic, lysine-containing peptides are able to form faint, stained precipitates. About 20 μg peptide per an average band contained in a 3.5-mm3 gel volume can be detected by this technique. It is hypothesized that the dye crosslinks different peptide chains by binding to basic residues via its SO3− groups.

Human endostatin-derived synthetic peptides possess potent antiangiogenic properties in vitro and in vivo

Pharmacological control of the angiogenic process (i.e., the neovascularization necessary for the growth and progression of tumors and metastases) is considered to be one of the most promising approaches to antineoplastic therapy. Endostatin, a 20-kDa protein derived from collagen XVIII, is one of the first recently discovered endogeneous antiangiogenic substances, but its cell targets and mechanism(s) of action are still unknown. We thought it would be interesting to test whether shorter peptides derived from endostatin might preserve its antiangiogenic activity. Four synthetic peptides corresponding to the sequences 6-49 (I), 50-92 (II), 93-133 (III), and 134-178 (IV) of human endostatin were tested for their ability to inhibit endothelial cell proliferation, migration, and both in vitro and in vivo angiogenesis. Fragment I (and fragment IV in the tests performed) was found to be fully biologically active in all of the angiogenesis assays, and sometimes showed even greater potency and efficacy than full-length human endostatin itself.

Local delivery of a synthetic endostatin fragment for the treatment of experimental gliomas

OBJECTIVE:Endostatin is an anti-angiogenic agent that blocks matrix-metalloproteinase-2 and inhibits endothelial cell proliferation. Currently, endostatin is available through recombinant technology, which limits its broader use. In this study, a synthetic endostatin fragment (EF) was analyzed to determine its anti-angiogenic properties when locally delivered by controlled-release polymers and to establish its effect as a treatment for experimental gliomas. METHODS:Cytotoxicity of EF against 9L gliosarcoma and F98 glioma was determined in vitro. EF was loaded into polyanhydride-poly-(bis-[carboxyphenoxy-propane]-sebacic-acid) (pCPP:SA) polymers at increasing concentrations. Pharmacokinetics of the EF/polymer formulations were defined in vitro. Anti-angiogenic properties of the EF/polymer formulations were evaluated in the rat-cornea micropocket assay. Toxicity and efficacy of locally delivered EF polymers either alone or combined with systemic bischloroethylnitrosourea (carmustine) were determined in rats intracranially challenged with 9L gliosarcoma. RESULTS:EF showed scarce cytotoxicity against 9L and F98 in vitro. EF/pCPP:SA formulations showed sustained release by day 19. Mean corneal angiogenesis index 20 days after tumor implantation was 4.5 ± 0.7 for corneas implanted with 40% EF/pCPP:SA compared with controls (8.5 ± 1.3, P = 0.02). Intracranial efficacy studies showed that EF polymers alone did not prolong animal survival. Combination of 40% EF/pCPP:SA polymers with systemic bischloroethylnitrosourea (carmustine) prolonged survival (median survival of 44 d, P = 0.001) and generated 33% long-term survivors. CONCLUSION:Controlled-release polymers can effectively deliver a biologically active EF in a sustained fashion. EF inhibits angiogenesis in vitro and in vivo, and even though EF does not prolong survival as a single agent, it exhibits a synergistic effect when combined with systemic bischloroethylnitrosourea (carmustine) in the intracranial 9L gliosarcoma model.

Iodine stain for detection of peptides after isoelectric focusing

Iodine stain is used for the detection of peptides after isoelectric focusing has been developed. Ultrathin gels (240-360 micrometer) are cast and, after focusing, dried at 110 degrees C on a filter paper sheet. The paper-pasted gel is then exposed to iodine vapors for a few seconds to a few minutes, depending on the peptide load. White peptide zones are visible on a brown, uniform background. The reaction is fully reversible and can be used also for small-scale preparative purification of peptides. Better than 80% recoveries of peptide from the gel can be obtained by elution in 80% acetic acid.

Molecular Models of Acidic Peptides from Pea Bud Chromatin and Seminal Plasma. Divalent Cations-Mediated Interaction with DNA

Abstract Small acidic peptides have been isolated from biological fluids (blood and seminal plasma) and from chromatin of several tissues. Their biological activity is related to the control of cell growth and gene expression. This work is an approach to the study of peptide structure-function relationship. Purified fractions from seminal plasma and pea bud chromatin were subjected to fast ion bombardment mass spectrometry. The results obtained were analyzed according to biochemical characteristics of the peptides studied and some possible molecular models have been designed. Two of the proposed sequences were synthesized and their biological activity assayed in cells and cell-free systems. The results demonstrate that the synthetic peptides are able to bind to DNA in the presence of divalent cations (Mg2+, Fe2+, Cu2+) with consequent inhibition of DNA transcription.

Synthesis, characterization and stereoselective catalytic oxidations of chelated deuterohaemin-glycyl-L-histidine complexes

Abstract The new complexes deuterohaemin-2(18)-glycyl-L-histidine methyl ester (DH-GH) and deuterohaemin-2,18-bis(glycyl-L-histidine) dimethyl ester (DH-bis(GH)) have been obtained by condensation of glycyl-L-histidine methyl ester on the propionic acid side-chains of deuterohaemin. The five-coordinated complex DH-GH, where the imidazole group of the histidine residue acts as the iron(III) axial ligand, is catalytically active in the oxidation of phenolic compounds by hydrogen peroxide, while the six-coordinated DH-bis(GH) complex, as expected, is a very poor catalyst in the same reaction. The kinetics of the catalytic oxidations of the L and D isomers of tyrosine (Tyr) and tyrosine methyl ester (Tyr-OMe) by the system DH-GH/H2O2 have been investigated in detail. The reactions occur with unexpected stereoselectivity, suggesting that the phenolic substrates are capable of interacting with the catalyst from the proximal side, which contains the chiral histidine centre. Analysis by molecular mechanics (MM) and molecular dynamics (MD) calculations indicates that the mode of substrate-haemin catalyst interaction brings the phenol group of the tyrosines to approach laterally the porphyrin macrocycle. This disposition is reminiscent of the aromatic substrate mode of interaction in peroxidases, where the electron transfer processes occur at the haem edge.

Analytical and preparative isoelectric focusing of peptides in immobilized pH gradients

A new method for peptide analysis and purification is described, based on isoelectric focusing in immobilized pH gradients. On the analytical scale, the peptide zones can now be revealed by any stain for primary and secondary amino groups (e.g. ninhydrin, fluorescamine, dansyl chloride) since the buffering species, unlike conventional carrier ampholytes, contain only carboxyl and tertiary amino groups. For preparative purposes, conditions have been described to remove most contaminants (e.g. unreacted monomers, non-cross-linked, short polyacrylamide chains) from the gel matrix before the electrophoretic run. However, ca. 2% of the gel dry mass is still present as extractable material. The focused peptides can be recovered in high yields (ca. 90%) with a fairly high degree of purity (75%), the contaminants being mostly components eluted from the polyacrylamide gel.

Inhibitory activity of a synthetic pentapeptide on leukaemic myelopoiesis both in vitro and in vivo in rats

The synthetic pentapeptide pGlu‐Glu‐Asp‐Cys‐Lys has recently been proposed as the active component of a granulocyte‐derived inhibitor of normal haematopoiesis. We investigated its biological activity on leukaemic myelopoiesis both in vitro and in vivo in rats. Three different human permanent myeloid leukaemic cell lines (HL60, KG1, ML3) and a rat transplantable acute myeloid leukaemia (Shay leukaemia) were studied. Neither HL60 nor KG1 were sensitive to the peptide whereas a consistently reproducible inhibition of 3H‐TdR uptake was observed in ML3 cells. This effect was not due to a unspecific toxic action on target cells and was spontaneously reversible. When injected i.p. twice daily at an appropriate concentration in rats bearing Shay leukaemia, the peptide caused a significant increase in survival. Our results therefore indicate that the synthetic pentapeptide studied inhibits not only normal but also leukaemic myelopoiesis.

Haem–peptide complexes. Synthesis and stereoselective oxidations by deuterohaemin-L-phenylalanyl-poly-L-alanine complexes

Deuterohaemin-L-histidine methyl ester—peptide complexes have been obtained by covalently linking L-histidine methyl ester and the peptides Ala-Ala-Phe-Ala-Ala-Ala-Ala-Ala-Ala-Ala (compound 3) or Ala-Ala-Ala-Phe-Ala-Ala-Ala-Ala-Ala-Ala (compound 4) on the propionic acid side chains. The spectroscopic properties of 3 and 4 and the imidazole binding equilibria indicate that folding of the peptide chain on the opposite part of the porphyrin plane with respect to that occupied by the bound histidine side-arm reduces in the order 3 > 4 the accessibility of exogenous ligands to the sixth co-ordination position of the iron atom, probably through some stacking interactions between the phenylalanine residue of the peptide and the porphyrin ring in the case of 3. This arrangement has marked consequences on the stereoselectivity observed in a model peroxidase reaction using L- or D-tyrosine methyl ester as substrates and tert-butyl hydroperoxide as oxidant in dichloromethane–trifluoroethanol (9:1), that have been interpreted in terms of the interaction between the chiral substrates and the peptide chains of the deuterohaemin complexes.

Synthesis, ligand binding and biomimetic oxidations of deuterohaemin modified with an undecapeptide residue

Deuterohaemin [(3,7,12,17-tetramethylporphyrin-2,18-dipropionato)iron(III)] has been covalently linked to the undecapeptide Ala-Phe-Ser-Phe-Glu-Ala-Gln-Gly-Gly-Leu-Ala at one of the propionic acid side chains. The binding equilibria between the resulting deuterohaemin-undecapeptide complex and imidazole occur in two steps; the first molecule of the ligand binds with high affinity (K1= 1500 dm3 mol–1), while for the second molecule the affinity is markedly lower (K2= 220 dm3 mol–1), indicating that folding of the peptide chain in the monoimidazole adduct severely limits the accessibility of the sixth iron co-ordination position. The complex exhibits both catalase and peroxidase activity towards reducing substrates in the presence of hydrogen peroxide. In catalytic sulphoxidations of a series of para-substituted thioanisoles, p-XC6H4SMe, by hydrogen peroxide a good correlation has been found between the relative rates and the Hammett σp values, indicating that direct oxygen transfer from the active oxoiron species to the sulphide is probably operative. The kinetics of the catalytic oxidation of tyrosine by hydrogen peroxide in the presence of the complex, producing the oxidative coupling dimer o,o′-dityrosine, was also studied. It is consistent with a mechanism involving the initial binding of the phenolic substrate to the active catalyst to form an intermediate complex, and its subsequent breakdown in the rate-determining step of the catalytic cycle. The results obtained in the biomimetic oxidations are compared with those of the corresponding peroxidase-catalysed reactions.