René Knecht

Separation, purification, and sequence identification of TGF-β1 and TGF-β2 from bovine milk

Cox and Bürk (Eur. J. Biochem., 1991) reported the partial characterization of Milk Growth Factor (MGF) which stimulated the migration of fibroblasts. We have fractionated the partially purified sample by RP-HPLC and obtained the separation of two peaks of activity. The two active components were isolated as pure MGF-a and MGF-b by RP-HPLC and preparative SDS-PAGE. The purified MGF-a, consisting of a single band by gel electrophoresis and a single peak on an HPLC reversed-phase C-4 column, has the same specific activity as TGF-β2 in the fibroblast migration assay. MGF-a was digested by endoprotease Asp-N and the cleaved peptides were analyzed by Edman degradation and plasma desorption mass spectrometry (PDMS). The whole sequence of MGF-a determined by automated sequenator and PDMS of S-pyridylethylated protein and selected fragments was found to be identical to that of TGF-β2. MGF-b protein mixture separated by SDS-PAGE was electrophoretically transferred onto a Biometra Glassybond membrane, and the blotted MGF-b protein was directly sequenced on an automated sequenator. The identified 29 amino acids sequence of MGF-b was identical to the amino-terminal sequence of TGF-β1. Our study demonstrates that MGF is composed of both TGF-β1 and TGF-β2. TGF-β2 (85%) is the predominant form.

Direct analysis of the disulfide content of proteins : methods for monitoring the stability and refolding process of cystine-containing proteins

So far, the cystine (disulfide) content of proteins has been routinely determined by indirect methods, i.e., cystines are first converted to more stable half-cystines prior to analysis. We present a study which demonstrates that the cystine content can be directly and accurately analyzed at low picomole to femtomole levels. The method involves (a) the employment of a vacuum during sample hydrolysis which permits quantitative recovery of cystine, and (b) the dabsyl chloride precolumn derivatization method which yields stable DABS-cystine that is subsequently analyzed by HPLC. Direct analysis of the cystine residue is important in numerous areas of protein research. It allows, by a single analysis, simultaneous determination of the sulfhydryl (cysteine, as S-carboxymethyl derivative) and disulfide (cystine) contents. The technique can be used to follow the refolding process of fully reduced, cystine-containing proteins. Direct analysis of the cystine content also serves to monitor the extent of inactivation of cystine-containing proteins caused by alkaline pH and heat treatments. In this mode of protein inactivation, cystines are selectively destroyed and converted to lanthionine and lysinoalanine. Both the decrease of cystine and the recoveries of lanthionine and lysinoalanine can be simultaneously evaluated by the proposed method. Examples of these applications are presented here.

Sequence determination of eglin c using combined microtechniques of amino acid analysis, peptide isolation, and automatic Edman degradation

The complete amino acid sequence of a proteinase inhibitor, eglin c (Mr 8100), has been determined with less than 150 micrograms of the protein using the following microtechniques: (a) amino acid analysis with a low-nanogram amount of protein hydrolysate using dimethylaminoazobenzene sulfonyl chloride, (b) peptide isolation at the picomole level using the dimethylaminoazobenzene isothiocyanate (DABITC) precolumn derivatization method, and (c) automatic Edman degradation. One amino acid residue has been corrected for the previously reported sequence. The Contribution of each technique to the microsequencing is discussed. In addition, a new high-performance liquid chromatography system that gives a complete baseline separation of all phenylthiohydantoin-amino acids is described.

Isolation and purification of novel hirudins from the leech Hirudinaria manillensis by high-performance liquid chromatography.

The isolation and purification of novel hirudins from a crude extract of the leech Hirudinaria manillensis and their analytical characterization are reported. Initial purification by gel permeation chromatography on Sephadex G50 and anion-exchange chromatography on Q Sepharose fast-flow removed most contaminants and yielded a highly active extract. Two isohirudins (designated hirudin P6 and P18) were isolated and purified by successive reversed-phase high-performance liquid chromatography on silica-based stationary phases and anion-exchange chromatography on Mono Q. The final products were characterized by reversed-phase high-performance liquid chromatography, 252Cf plasma desorption time-of-flight mass spectrometry and capillary zone electrophoresis. The molecular masses determined by 252Cf plasma desorption mass spectrometry were 7416 dalton for hirudin P6 and 7199 dalton for hirudin P18.

High Sensitivity Amino Acid Analysis Using DABS-Cl Precolumn Derivatization Method

Precolumn derivatization of amino acids and subsequent HPLC analysis of amino acid derivatives have provided a viable alternative to the conventional analyzer for high sensitivity amino acid analysis. Several reagents are available for this new technique. All amino acid derivatives of DNS-C1 [1, 2], OPA [3–5], NDB-F [6, 7], PITC [8, 9] and DABS-C1 [10–17] can be reproducibly prepared and detected at the low picomole level. Among them, the DABS-C1 method is unique in two ways, (a) DABS-amino acids are detected in the visible region. At low picomole level, a stable baseline can be readily obtained with a large variety of solvent and gradient systems, (b) DABS-amino acids, like DNS-amino acids (with their sulfonamide bonds) are comparably the most stable derivatives. They can be left at room temperature for a period of up to two months without any appreciable degradations. This high stability is a prerequisite for the reliable quantitative analysis of amino acid derivatives.

Isolation and Sequence Determination of Polypeptides at the Picomole Level

Increase of sensitivity for protein sequencing depends on the development of micro-methods for amino acid analysis, peptide isolation and sequence determination. While sensitive automatic methods for both amino acid analysis (1,2) and sequence determination (3) are available, there is no straight forward method for peptide isolation. Resolution of a complex peptide mixture usually requires the successive use of combined chromatographic methods (4). The procedure is time-consuming and costly with regard to material since peptide loss occurs with each purification step. Therefore, an ideal peptide isolation method should be not only sensitive but also alone powerful enough to completely resolve peptide mixtures. We have developed new techniques to meet these requirements by precolumn derivatization of amino acids and peptide with chromophoric reagents. The methods used and the results obtained are summarized and discussed in this paper.