Disulfide structure of the heparin binding domain in vascular endothelial growth factor: characterization of posttranslational modifications in VEGF.

Preparations of recombinant human vascular endothelial growth factor (VEGF165) expressed in Chinese hamster ovary (CHO) cells and Escherichia coli were compared using a variety of analytical methods. Amino terminal sequence analyses of both the CHO- and E. coli-derived VEGF165 confirmed the predicted amino terminal sequence for VEGF165, although the CHO VEGF165 exhibited a heterogeneous amino terminus with sequences beginning at Ala-1 (76%), Pro-2 (4%), Ala-4 (13%), and Glu-5 (7%). Tryptic digests of reduced and carboxymethylated CHO- and E. coli-derived VEGF165 were examined by LC/MS analyses, indicating equivalent primary structure, except for the glycosylation at Asn-75 in the CHO-derived VEGF165. The N-linked carbohydrate in the CHO-derived VEGF165 was determined to be a complex fucosylated biantennary structure. The data obtained from LC/MS analysis and amino terminal sequence analysis of VEGF165 confirmed 98% of the primary structure. Disulfide linkages for the eight cysteine residues in the carboxyl terminal heparin binding domain were assigned by amino terminal sequencing of fragments produced by tryptic digests of each native molecule. The following disulfides have been identified for both CHO- and E. coli-derived VEGF165: Cys-117 and Cys-135, Cys-120 and Cys-137, Cys-139 and Cys-158, plus Cys-146 and Cys-160. Plasmin cleavage of VEGF165 yields an N-terminal homodimeric VEGF110 and a 55-amino-acid carboxyl terminal domain. VEGF110 was resistant to further proteolytic or chemical digestion such that the disulfide linkages were not elucidated. The 55-amino-acid carboxyl terminal region of VEGF165 appears to be a unique heparin binding domain with no known protein homology.

The use of t-butyl hydroperoxide as a probe for methionine oxidation in proteins.

The susceptibility of native recombinant interferon gamma (rIFN-gamma, Actimmune) and recombinant tissue-type plasminogen activator (rt-PA, Activase) to methionine oxidation when treated with the oxidizing agent t-butyl hydroperoxide (TBHP) was investigated. The results showed that two of the five methionine residues in rIFN-gamma were susceptible to oxidation by TBHP, while three of the five methionines in rt-PA were found to be oxidizable. The oxidized methionine residues were found to be in the sulfoxide [Met(O)] form, and no other residue(s) appeared to be modified during the TBHP treatment. These results also showed that during treatment of a native protein with TBHP only the exposed methionine residues were oxidized. The biological activity of both molecules were unaffected by the treatment with TBHP. A comparative study between TBHP and hydrogen peroxide (H2O2) demonstrated that H2O2 was also a methionine-specific oxidizer. However, this study also showed that H2O2 was not able to distinguish between exposed and buried methionine residues, as significant portions of all five methionine residues in native rIFN-gamma were oxidized by treatment with H2O2. TBHP should be useful for identifying surface methionine residues in a protein of unknown structure and a valuable reagent for methionine oxidation in pharmaceutical stability studies.

Study of the primary structure of recombinant tissue plasminogen activator by reversed-phase high-performance liquid chromatographic tryptic mapping.

Two high-resolution tryptic maps have been developed for recombinant tissue plasminogen activator (rt-PA) that separate the expected 51 tryptic peptides. The trypsin digestion was performed after reduction and S-carboxymethylation of the protein. The high-performance liquid chromatographic separation of the tryptic peptides used a Nova-Pak C18 (5 microns) column with a mobile phase that contained 0.1% aqueous trifluoroacetic acid (TFA) or 50 mM sodium phosphate (pH 2.85) and a linear gradient of acetonitrile. A TFA solvent system was also used for re-purification and for characterization of the peptides isolated from the phosphate-based separation. All of the isolated peptides had compositions consistent with the sequence proposed for rt-PA. The identities of the glycopeptides were confirmed by lectin chromatography on concanavalin A-Sepharose. The mixture of tryptic peptides was also treated with endo-beta-N-acetylglucosaminidase H and peptide:N-glycosidase F to locate the position of either high mannose or complex oligosaccharides. These studies demonstrated that a high mannose oligosaccharide is attached to Asn-117 while complex carbohydrate side-chains are attached to Asn-184 and Asn-448. The residue Asn-184 is the site of optional glycosylation that results in the formation of two rt-PA variants that contain either two or three oligosaccharides.

Application of high-pressure liquid chromatography and thermal energy analyzer to analysis of trinitroglycerin and its metabolites in blood.

A highly selective and sensitive analytical procedure for the determination of trinitroglycerin and four metabolites in whole blood was developed. Trinitroglycerin and its metabolites were extracted from whole blood with ethyl acetate and analyzed by high-pressure liquid chromatography using the thermal energy analyzer detector. Linearity of response was observed over the 1-1000-ng range. The applicability of this method to the analysis of whole blood from dogs orally dosed with trinitroglycerin is described.