Leucine-rich 3,1-S-α2 -glycoprotein, process for isolating it and its use

Claim 2

Glycoprotein isolated from human serum, characterized by the following parameters: (a) sedimentation constant in the ultracentrifuge 3.1 S±0.2; (b) molecular weight of 49,000±4,000; (c) electrophoretic mobility in the range of the α 2 -globulins of the human serum; (d) isoelectric range pH 3.8-4.1; (e) extinction coefficient E 1 cm 1% , 280 nm=7.0±0.2; (f) content of about 77%±2% of α-amino-acids and about 23%±2% of carbohydrates, and of said amino acids and carbohydrates, the major fractions being present as follows: ______________________________________ Mole % of the amino-acid pro- Variation Amino Acids portion coefficient % ______________________________________ Aspartic acid 11.45 1.49 Serine 6.43 2.36 Glutamic acid 10.72 2.51 Proline 7.04 4.43 Glycine 7.32 2.31 Alanine 6.65 1.97 Leucine 22.05 5.79 ______________________________________ Carbohydrate Residues % by weight ______________________________________ Galactose 4.1 ± 0.3 Mannose 3.8 ± 0.3 N-Acetylhexosamine 8.1 ± 0.5 N-Acetylneuraminic acid 6.6 ± 0.4 ______________________________________ (g) specific immunological reactions with antibodies directed against the glycoprotein.

Claim 3

Glycoprotein isolated from human serum, characterized by the following parameters: (a) sedimentation constant in the ultracentrifuge 3.1 S±0.2; (b) molecular weight of 49,000±4,000; (c) electrophoretic mobility in the range of the α 2 -globulins of the human serum; (d) isoelectric range pH 3.8-4.1; (e) extinction coefficient E 1 cm 1% , 280 nm=7.0±0.2; (f) content of about 77%±2% of α-amino-acids and about 23%±2% of carbohydrates, said amino acids and carbohydrates being present as follows: ______________________________________ Mole % of the amino-acid pro- Variation Amino Acids portion coefficient % ______________________________________ Lysine 3.87 5.90 Hystidine 2.23 11.28 Arginine 4.65 7.68 Aspartic acid 11.45 1.49 Threonine 3.87 6.26 Serine 6.43 2.36 Glutamic acid 10.72 2.51 Proline 7.04 4.43 Glycine 7.32 2.31 Alanine 6.65 1.97 1/2 Cystine 1.21 9.83 Valine 4.48 3.78 Methionine 0.75 5.81 Isoleucine 1.29 3.16 Leucine 22.05 5.79 Tyrosine 0.81 19.53 Phenylalanine 3.23 1.39 Tryptophane 1.60 11.69 ______________________________________ Carbohydrate Residues % by weight ______________________________________ Galactose 4.1 ± 0.3 Mannose 3.8 ± 0.3 Glucose 0 Fucose 0.3 ± 0.1 Xylose 0 N-Acetylhexosamine 8.1 ± 0.5 N-Acetylneuraminic acid 6.6 ± 0.4 ______________________________________ (g) specific immunological reactions with antibodies directed against the glycoprotein.

Claim 4

Glycoprotein isolated from human serum, characterized by the following parameters: (a) sedimentation constant in the ultracentrifuge 3.1 S±0.2; (b) molecular weight of 49,000±4,000; (c) electrophoretic mobility in the range of the α 2 -globulins of the human serum; (d) isoelectric range pH 3.8-4.1; (e) extinction coefficient E 1 cm 1% , 280 nm=7.0±0.2; (f) content of about 77%±2% of α-amino-acids and about 23%±2% of carbohydrates, said amino acids and carbohydrates being present as follows: ______________________________________ Mole % of the amino-acid pro- Variation Amino Acids portion coefficient % ______________________________________ Lysine 3.87 5.90 Hystidine 2.23 11.28 Arginine 4.65 7.68 Aspartic acid 11.45 1.49 Threonine 3.87 6.26 Serine 6.43 2.36 Glutamic acid 10.72 2.51 Proline 7.04 4.43 Glycine 7.32 2.31 Alanine 6.65 1.97 1/2 Cystine 1.21 9.83 Valine 4.48 3.78 Methionine 0.75 5.81 Isoleucine 1.29 3.16 Leucine 22.05 5.79 Tyrosine 0.81 19.53 Phenylalanine 3.23 1.39 Tryptophane 1.60 11.69 ______________________________________ Carbohydrate Residues % by weight ______________________________________ Galactose 4.1 ± 0.3 Mannose 3.8 ± 0.3 Glucose 0 Fucose 0.3 ± 0.1 Xylose 0 N-Acetylhexosamine 8.1 ± 0.5 N-Acetylneuraminic acid 6.6 ± 0.4 ______________________________________ (g) specific immunological reactions with antibodies directed against the glycoprotein.

Claim 5

Process for the isolation of the protein claimed in claim 1, wherein a solution containing said protein is subjected to a combination of the following steps to obtain it in pure form, as demonstrated immunologically whereafter, in each step, the fraction containing said protein is isolated, and the end product recovered after the last step: (a) addition of a water-soluble salt of an acridine base up to a concentration of 0.01 moles per liter at a neutral or weakly alkaline pH to precipitate accompanying proteins; (b) addition of a neutral salt until the protein precipitates; (c) adsorption of the protein on an anion exchanger and elution from it; (d) molecular sieve fractionation and isolation of proteins having a molecular weight of between 45,000 and 55,000; and (e) adsorption of accompanying proteins with water-insoluble calcium phosphate-hydrate.

Claim 6

Process for the isolation of the protein claimed in claim 1, wherein a solution containing said protein is subjected to a combination of the following steps to obtain it in pure form, as demonstrated immunologically whereafter, in each step, the fraction containing said protein is isolated, and the end product recovered after the last step: (a) addition of a neutral salt until the protein precipitates; (b) addition of an organic solvent until accompanying proteins are precipitated; (c) addition of a water-soluble salt of an acridine base until precipitation of accompanying proteins; (d) addition of an organic acid until precipitation of accompanying proteins; (e) heating to a temperature of between 95° to 100° C. until precipitation of accompanying proteins; (f) reduction of the electrolyte content until precipitation of accompanying proteins; (g) molecular sieve fractionation and isolation of the proteins having a molecular weight of between 45,000 and 55,000; (h) adsorption of the protein on an anion exchanger and elution from it; (i) electrophoresis and isolation of the α 2 -zone of human serum; and (k) adsorption of accompanying proteins with water-insoluble calcium phosphate-hydrate.

Claim 7

Glycoprotein isolated from human serum, prepared according to the process of claim 4 and further characterized by the following parameters: (a) sedimentation constant in the ultracentrifuge 3.1 S±0.2; (b) molecular weight of 49,000±4,000; (c) electrophoretic mobility in the range of the α 2 -globulins of the human serum; (d) isoelectric range pH 3.8-4.1; (e) extinction coefficient E 1 cm 1% , 280 nm=7.0±0.2; (f) content of about 77%±2% of a α-amino-acids and about 23%±2% of carbohydrates, and of said amino acids and carbohydrates, the major fractions being present as follows: ______________________________________ Mole % of the amino-acid pro- Variation Amino Acids portion coefficient % ______________________________________ Aspartic acid 11.45 1.49 Serine 6.43 2.36 Glutamic acid 10.72 2.51 Proline 7.04 4.43 Glycine 7.32 2.31 Alanine 6.65 1.97 Leucine 22.05 5.79 ______________________________________ Carbohydrate Residues % by weight ______________________________________ Galactose 4.1 ± 0.3 Mannose 3.8 ± 0.3 N-Acetylhexosamine 8.1 ± 0.5 N-Acetylneuraminic acid 6.6 ± 0.4 ______________________________________ (g) specific immunological reactions with antibodies directed against the glycoprotein, which demonstrates the presence of said isolated glycoprotein.