Charles B. Glaser

Primary structure of neocarzinostatin, an antitumor protein.

The antitumor protein neocarzinostatin is an acidic single-chain molecule, cross-linked by two disulfide bridges, and consists of 109 amino acid residues. Complete disulfide bond reduction and S-carboxymethylation was achieved in liquid ammonia. Sequence determination of five tryptic fragments led to the proposed primary structure.

Structure of the antitumor protein neocarzinostatin. Amino acid sequence.

Abstract The amino acid sequence of the antitumor protein neocarzinostatin has been proposed, as shown in Fig. 7, by sequence analysis of tryptic peptides and fragments obtained by digestion of the protein with chymotrypsin, pepsin, acid protease, and dilute hydrochloric acid. Neocarzinostatin consists of 109 amino acid residues and its molecular weight, calculated from the proposed structure, is 10,717.

The isolation of alpha-1-protease inhibitor by a unique procedure designed for industrial application

Abstract Individuals who are congenitally deficient in the human plasma protein α 1 -protease inhibitor ( α 1 PI, which is also called α 1 -antitrypsin) usually develop chronic obstructive lung disease as a consequence of improperly regulated granulocyte elastase. In this report, a unique, facile one- or two-step method is presented for the large-scale isolation of α 1 PI for potential therapeutic use. The method takes advantage of the unusual disulfide bond in α 1 PI, which consists of a single cysteine residue in the polypeptide chain bound to a free pendant cysteine. In contrast to other circulating plasma proteins, the disulfide bridge in α 1 PI does not add to its structural stability. Therefore, if an α 1 PI-containing solution of plasma proteins is precipitated out in the presence of reductant, much more extensive separation of contaminating proteins will be achieved than in the absence of such reductant. We have used Cohn Fraction IV-1, a relatively unused side product in albumin and gamma globulin production, as our starting material. After activation of the α 1 PI in basic media, partial purification is achieved with successive additions of Aerosil (a fumed silica), dithiothreitol, and ammonium sulfate. From 90 to 95% of the contaminating proteins are precipitated by this single procedure, resulting in a product that is ∼70% pure. DEAE-cellulose chromatography can be used as an additional purification step, and this results in a product that is nearly homogenous. Overall yield is ∼45%. The method is simple, inexpensive, and reproducible and is directly applicable to large-scale industrial processing.

Structure of the antitumor protein neocarzinostatin: Purification, amino acid composition, disulfide reduction, and isolation and composition of tryptic peptides☆

Abstract The antitumor protein neocarzinostatin has been purified by repeated CM-cellulose chromatography and Sephadex G-50 gel filtration. The protein possesses 109 amino acid residues in a single chain, crosslinked by two disulfide bonds. Reduction and S -alkylation could not be accomplished in aqueous solution and required the use of dithiothreitol in liquid ammonia, followed by treatment with alkyl chloride. Tryptic digestion of (tetra- S -carboxymethyl)neocarzinostatin afforded five tryptic fragments which were fractionated by preparative paper chromatography and high-voltage paper electrophoresis, purified by Sephadex gel filtration and characterized by amino acid and amino end-group analysis. The total number of amino acid residues of these fragments account for the 109 residues of neocarzinostatin.

Affinity chromatography of α-1-protease inhibitor using Sepharose-4B-bound anhydrochymotrypsin

Abstract Sepharose 4B-bound bovine anhydrochymotrypsin (AnhCT), a catalytically inactive form of chymotrypsin, was shown to be effective for retaining active α-1-protease inhibitor (α-1-PI, also α-1-antitrypsin) from human plasma, while showing no measurable affinity for oxidized or protease complexed α-1-PI, or for most other plasma proteins. α-1-PI eluted from this resin with 0.1 m chymostatin retained full activity against trypsin, chymotrypsin, and elastase. In addition to α-1-PI, AnhCT-Sepharose binds a limited number of other plasma proteins. Using monospecific antisera to plasma protease inhibitors, one of these proteins was identified as inter-α-trypsin inhibitor, and it was recoverable in active form. Therefore, an AnhCT-Sepharose 4B resin has been demonstrated to be of value for isolating active forms of α-1-PI from solutions, and may also be useful for the isolation of inter-α-trypsin inhibitor.