Direct synthesis and characterisation of multi-dendritic peptides for use as immunogens.

The direct synthesis and subsequent rapid characterisation of multiple antigen peptides (MAPs) for use as immunogens has presented difficulties, partly because of the formation of incomplete or truncated peptide sequences during the synthetic procedure. Therefore, many researchers have resorted to ligation procedures for the synthesis of MAP constructs. This article describes a method to improve the yield of MAP constructs by direct synthesis methods, as well as a general procedure that enables easier characterisation of the synthetic products. In particular, during the synthesis of MAP constructs, a capping procedure was introduced after each amino acid coupling step, thus improving significantly the yield of the desired multi-dendritic peptidic immunogens. Through the use of this capping procedure, problems arising from the incomplete amino acid residue coupling at the point of synthesis were minimised, and any deletion peptides which formed could be eliminated more readily during the subsequent purification procedures. In addition, previous difficulties in purification and characterisation of MAP construct by, e.g. electrospray mass spectroscopy (ES-MS), often led to the multi-dendritic peptidic immunogens being used without full characterisation after dialysis and recovery of the product(s). This article describes an enzymatic (tryptic) digestion method with the MAP construct, followed by characterisation of the enzymatic digest by reversed phase high-performance liquid chromatography-ES-MS. With this method, fragments of the MAP construct cleaved at specific amino acid residue sites (e.g. lysine or arginine) within the sequence of the parent peptide can be readily determined and the kinetics of the digestion easily followed. This enzymatic digestion procedure thus provides a facile approach to confirm that all of the multi-dendritic arms of the purified MAP construct have been equivalently elongated during the peptide synthesis and that consequently the purified construct structure contains the correct peptide sequence.

Application of a sensitive HPLC-based fluorometric assay to determine the sialic acid content of human gonadotropin isoforms.

The human pituitary gonadotropins, follitropin (hFSH) and lutropin (hLH) are glycoproteins which are microheterogeneous in terms of their charge and molecular size, as well as their in vitro and in vivo bioactivities. The aim of this study was to determine the contribution of variations in sialic acid (N-acetyl neuraminic acid) content to the structural heterogeneity of these glycoproteins. Sialic acid (Neu5Ac) was released by partial acid hydrolysis (0.1 M TFA, 80 degrees C, 1 h) and derivatised with the fluorescent label DMB (1,2-diamino-4,5-methylenedioxybenzene) in conjunction with an internal standard (N-glycoyl-neuraminic acid). The derivatives were then separated by reversed-phase HPLC. This method allowed quantitation of the sialic acid content over a range of 5-100 pmol with between assay variation of < 6% for sialic acid released from approximately 100 ng (3 pmol) of hFSH or hLH. Comparison of the sialic acid contents of standard sialylated glycoproteins by either DMB-derivatisation or high-performance anion-exchange chromatography with pulsed amperometric detection yielded similar results, confirming the reliability of the fluorescence detection method. The sialic acid contents of 9 hFSH isoforms varied between 1.5-13.7 mol Neu5AC/mol FSH, whilst a range of 1.1-9.1 mol Neu5AC/mol LH was observed for 12 hLH isoforms. The sialic acid content of the hFSH isoforms was also observed to be related to the hormonal specific activity in a radioreceptor assay, confirming that alterations in the carbohydrate structure can influence the FSH-receptor interaction. In contrast, the sialic acid content of the hLH isoforms was found to be not related to specific activity at the receptor level.

Application of reversed-phase high-performance liquid chromatography to the separation of apolipoproteins A-IV, A-I and E from rat high-density lipoprotein.

Apolipoproteins A-IV, A-I and E from rat high-density lipoprotein (HDL) were successfully purified by reversed-phase high-performance liquid chromatography (RP-HPLC), using a method which we have previously developed for the separation of apolipoproteins A-IV, A-I and E from human lymph chylomicrons [T. Tetaz, E. Kecorius, B. Grego and N. Fidge, J. Chromatogr., 511 (1990) 147]. Since analytical-scale RP-HPLC indicated that the C apolipoproteins from rat HDL coeluted with both apo A-IV and apo A-I, delipidated rat HDL was first subjected to preparative-scale size-exclusion HPLC (HPSEC) on a Serva Si300 column, which effectively separated the C apolipoproteins from all but apolipoprotein E. Fractions from HPSEC which were enriched for apolipoproteins A-IV, A-I or E were directly applied to RP-HPLC on a TSK Phenyl-5PW column. This procedure yielded fractions containing apolipoproteins A-IV, A-I or E which were pure as assessed by N-terminal sequencing and silver staining of sodium dodecyl sulphate-polyacrylamide gels.

Separation of human apolipoproteins A-IV, A-I and E by reversed-phase high-performance liquid chromatography on a TSK Phenyl-5PW column.

A method has been developed for the rapid separation of the medium-molecular-weight apolipoproteins A-IV, A-I and E by high-performance liquid chromatography. Separations were achieved using a commercially available column of very low hydrophobicity (TSK Phenyl-5PW) in the reversed-phase mode rather than the conventional mode of hydrophobic interaction. Delipidated apolipoproteins were dissolved in 20 mM orthophosphoric acid (pH 2.3), applied to the column which was pre-equilibrated with the same buffer, and eluted with an increasing gradient of acetonitrile. Purified apolipoproteins were identified by a combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis, amino acid analysis and N-terminal sequence analysis. In one step the method can be used to separate the major human chylomicron apolipoproteins A-IV, A-I and E, following preliminary removal of apolipoprotein A-II and the C apolipoproteins by size-exclusion chromatography.

Characterization of a dipeptidyl aminopeptidase from bovine adrenal medulla.

A dipeptidyl aminopeptidase was partially purified from a supernatant fraction of bovine adrenal medulla by gel filtration and anion-exchange chromatography. From gel filtration, the apparent molecular weight of the enzyme was 68,100 and its pH optimum was 9.5. Its Km for hydrolysis of the synthetic substrate arginylarginine-beta-naphthylamide was 5.5 X 10(-6) M. The enzyme was inhibited by metal ion chelating agents and thiol blocking agents, suggesting the requirement for both a metal ion and an active cysteine residue for its activity. Several peptides were cleaved by the dipeptidyl aminopeptidase involving the sequential removal of dipeptides from the N-terminus. Biologically active peptides, such as leucine-enkephalin, methionine-enkephalin, and angiotensin II, were hydrolyzed by the dipeptidyl aminopeptidase although opioid peptides with a length greater than five amino acid residues were not susceptible to hydrolysis. Other peptides with a blocked N-terminus (neurotensin, bombesin) or a proline residue adjacent to a potential cleavage site (substance P) were not hydrolyzed. The ability of this dipeptidyl aminopeptidase to degrade certain neuropeptides suggests that it could be involved in neuropeptide degradation.