US 4,054,557 A · Growth promoting polypeptides and preparation method

Abstract

The application discloses (A) a growth-promoting polypeptide having molecular weight from about 5,000 to about 7,000 chromatographic mobility under electrophoresis from about 0.25 to about 0.37 relative to lysine at pH 5 and from about 0.17 to about 0.43 relative to aspartic acid at pH 7.5, terminal amino acid at its amino end being asparagine or aspartic acid, and respective amounts of aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, leucine, tyrosine, phenylalanine, lysine, arginine, trypophan, and cysteine or carboxymethyl cysteine, and also a couple of instances also methionine, isoleucine and histidine; and (B) the derivation of the polypeptides from blood serum or plasma or a plasma fraction by combined steps of (a) homogenizing the starting material in water, (b) admixing a solution of hydrochloric acid in ethanol thereby splitting the polypeptide from its carrier proteins and precipitating them, (c) adjusting the pH to 8.4 to provide a precipitate, (d) separating it, (e) adjusting the pH of the liquid residue to 3, (f) admixing a water-insoluble strongly acidic cation exchange matrix of sulfopropyl-oxygen-linked-substitued dextran cross-linked with epichlorhydrin at pH 3, (g) separating the gel matrix from the liquid, (h) eluting the polypeptide-containing material from the matrix with (x) ammonium acetate at pH 10 or (y) 0.75M ammonium bicarbonate; and (i) precipitating the polypeptide by admixing acetone.

Claims16

Dependent claims

A growth-promoting polypeptide derivable from mammalian blood plasma by a procedure which includes subjecting the plasma material to an acid, bond-splitting agent;

and having (i) molecular weight in the range of from about 5,000 to about 7,000 determined by gel filtration enhanced by using insulin of its known molecular weight as a standard for comparison, (ii) chromatographic mobility under electrophoresis relative to lysine at pH 5 of from about 0.25 to about 0.37 and relative to aspartic acid at pH 7.5 of from about 0.17 to about 0.43, and (iii) the terminal amino acid at its amino end is asparagine or aspartic acid, and also having the respective constitution provided by one of the following groups (a), (b), and (c) of combined amino acids, with each said acid being in its respectively noted molar content:
a. aspartic acid 4.1, threonine 4.3, serine 7.8, glutamic acid 5.1, proline 8.2, glycine 10.2, alanine 4.9, valine 4.0, methionine 0.3, isoleucine 1.3, leucine 2.1, tyrosine 0.6, phenylalanine 1.1, histidine 1.5, lysine 2.8, arginine 4.0, tryptophan 1.0, and carboxymethyl cysteine 1.0, per mol of said polypeptide;
b. aspartic acid 4.8, threonine 1.9, serine 3.5, glutamic acid 4.6, proline 7.3, glycine 7.1, alanine 7.0, valine 4.9, methionine 0.9, isoleucine 0.8, leucine 3.3, tyrosine 0.9, phenylalanine 2.2, histidine 3.8, lysine 2.5, arginine 4.6, tryptophan 1.0, and cysteine (determined as cysteic acid) 1.14, per mol of said polypeptide;
and
c. aspartic acid 5.2, threonine 4.0, serine 3.1, glutamic acid 8.1, proline 0.8, glycine 2.1, alanine 1.1, valine 1.8, leucine 1.0, tryosine 2.9, phenylalanine 1.0, lysine 3.8, arginine 1.3, tryptophan 1.0, and carboxymethyl cysteine 8.0, per mol of said polypeptide.

2 more elements

In the method of deriving a growth-promoting polypeptide having molecular weight in the range of from about 5,000 to about 7,000, chromatographic mobility under electrophoresis relative to lysine at pH 5 of from about 0.25 to about 0.37 and relative to aspartic acid at pH 7.5 of from about 0.17 to about 0.43, and the terminal amino acid at its amino end being asparagine or aspartic acid, from mammalian blood serum or plasma or a plasma fraction from which said polypeptide can be derived, the combination of steps comprising (a) homogenizing the Cohn et al. plasma fraction IV or fraction IV-6+7 with sufficient water and for a time sufficient to homogenize said fraction, (b) subjecting the resulting homogenate with a sufficient polypeptide-to-protein-linkage -bond-splitting amount of a mixture of about 1 part of concentrated hydrochloric acid in about forty parts of about 96% ethanol at about 0.degree. C for a time sufficient to split said polypeptide from its carrier proteins and to precipitate said proteins, (c) adjusting the pH of the reaction mixture to about 8.4 with the required amount of a compatible water-soluble alkali metal hydroxide, (d) separating the precipitate from the liquid vehicle, (e) adjusting the pH of said liquid vehicle to about 3 by cautiously admixing it with concentrated hydrochloric acid, (f) admixing the resulting acid liquid vehicle at about 0.degree. C with a polypeptide-adsorbing sufficient amount of a finely divided, water-insoluble strongly acidic cation exchange gel matrix constituted of a three-dimensional network of sulfopropyl-oxygen-linked-substituted dextran chains cross-linked with epichlorhydrin equilibrated with sodium chloride at about pH 3, for a time sufficient to adsorb said polypeptide;

(g) separating said water-soluble gel matrix with said adsorbed polypeptide from its surrounding liquid vehicle, (h) eluting the polypeptide containing material from said gel matrix with (x) about 0.2M ammonium acetate adjusted to pH 10 by addition of about 25% ammonium hydroxide, or (y) about 0.75M ammonium bicarbonate;
and (i) precipitating the crude polypeptide from the resulting eluate by admixing the latter at about -15.degree. C with about 4 times its volume of acetone.

Dependencies are inferred from the claim text.

Growth promoting polypeptides and preparation method

Life of this patent

Growth promoting polypeptides and preparation method

Life of this patent

Abstract

Claims16