CA2286971A1 - A method for the purification of natural phosphorylated peptides, a mixture of phosphopeptides thus obtained and the use thereof - Google Patents
A method for the purification of natural phosphorylated peptides, a mixture of phosphopeptides thus obtained and the use thereof Download PDFInfo
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Abstract
A method for the preparation of phosphorylated peptides from casein hydrolyzates which can produce a mixture of soluble peptides with a high degree of purity containing a high percentage of phosphoserine, is described. The method comprises adsorption of the casein hydrolyzate on alumina and elution with ammonium hydroxide. The purified mixture of phosphopeptides thus obtained is particularly useful in pharmaceutical compositions for promoting intestinal absorption of metal ions.
Description
r DESCRIPTION
The present invention relates to a method of preparing phosphorylated peptides from casein hydrolyzates which produces a mixture of soluble peptides with a high degree of purity, containing a high percentage of phosphoserine.
It is known that ~ milk casein and its derivatives contain phosphoserines which impart valuable physical, chemical, technological and physiological properties to the peptides present therein.
Some authors have shown that intestinal absorption of calcium increases as a result of the administration of casein in the rat and that the phosphopeptides of casein are responsible for this effect (Y.S. Lee, D. Noguchi and H. Naito, Brit. J. Nutr. 49, 67, 1983) .
Casein., phosphopeptides also increase calcium absorption in normal chickens and those suffering from rickets (H. M. Mykkanen and R.H. Wasserman, J. Nutr. 110, 2141, 1980).
It is therefore of interest to obtain casein phosphopeptides in substantially pure form by simple and inexpensive methods.
The methods used up to now for the purification of phosphopeptides from tryptic casein hydrolyzates provide for precipitation with the use of a calcium or Fe3+ salt (EP-A-0 090 406 and JP-59-159793) or passage over activated carbon or over cation-exchange resin (JP-59-159792).
It is known that aluminium (A1203) produced by thermal dehydration of aluminium hydroxide, in the form of a powdery, porous material, generally in particles of from 40 to 250 mesh, can be used for the specific immobilization of phosphoric acid (R-P03H2) and as a substrate for rendering phosphorylated biomolecules insoluble in . accordance with affinity chromatography principles; these uses have been described and discussed by Coletti-Previero and Previero (Analyt. Biochem.
180, 1-10, 1989).
EP-A-0 218 506 describes a method for the adsorption of monosubstituted derivatives of phosphoric acid on alumina and their subsequent elution by means of a phosphate buffer. The specific applications comprise the fixing of phosphoderivatives on alumina at ambient temperature and at a pH of between 2.0 and 8.5 in order to isolate and determine these phosphoderivatives in biological media (for example, to render phosphorylated enzymes insoluble).
It has been found that alumina can advantageously be used for the purification of phosphopeptides from casein hydrolyzates, producing a mixture of soluble peptides with a high degree of purity, containing a large quantity of phosphoserine.
A first subject of the invention is therefore a method for the selective purification of phosphopeptides from hydrolyzed casein, characterized in that it comprises adsorption of the casein hydrolyzate on alumina and elution of the selectively-adsorbed phosphopeptides with ammonium hydroxide.
Another subject of the invention is a mixture of purified phosphopeptides which can be produced by the above-mentioned selective adsorption and elution method.
The method according to the invention has considerable advantages such as speed, a good purification yield, and an absence of contaminants in the peptides obtained. The use of ammonium hydroxide for the elution of the phosphopeptides constitutes a further improvement since it avoids contaminating the peptides with the phosphorus contained in the buffer and the eluent, and because the eluent can easily be removed in the form of ammonia in the subsequent peptide lyophilization process.
1 ' . . , .
The present invention relates to a method of preparing phosphorylated peptides from casein hydrolyzates which produces a mixture of soluble peptides with a high degree of purity, containing a high percentage of phosphoserine.
It is known that ~ milk casein and its derivatives contain phosphoserines which impart valuable physical, chemical, technological and physiological properties to the peptides present therein.
Some authors have shown that intestinal absorption of calcium increases as a result of the administration of casein in the rat and that the phosphopeptides of casein are responsible for this effect (Y.S. Lee, D. Noguchi and H. Naito, Brit. J. Nutr. 49, 67, 1983) .
Casein., phosphopeptides also increase calcium absorption in normal chickens and those suffering from rickets (H. M. Mykkanen and R.H. Wasserman, J. Nutr. 110, 2141, 1980).
It is therefore of interest to obtain casein phosphopeptides in substantially pure form by simple and inexpensive methods.
The methods used up to now for the purification of phosphopeptides from tryptic casein hydrolyzates provide for precipitation with the use of a calcium or Fe3+ salt (EP-A-0 090 406 and JP-59-159793) or passage over activated carbon or over cation-exchange resin (JP-59-159792).
It is known that aluminium (A1203) produced by thermal dehydration of aluminium hydroxide, in the form of a powdery, porous material, generally in particles of from 40 to 250 mesh, can be used for the specific immobilization of phosphoric acid (R-P03H2) and as a substrate for rendering phosphorylated biomolecules insoluble in . accordance with affinity chromatography principles; these uses have been described and discussed by Coletti-Previero and Previero (Analyt. Biochem.
180, 1-10, 1989).
EP-A-0 218 506 describes a method for the adsorption of monosubstituted derivatives of phosphoric acid on alumina and their subsequent elution by means of a phosphate buffer. The specific applications comprise the fixing of phosphoderivatives on alumina at ambient temperature and at a pH of between 2.0 and 8.5 in order to isolate and determine these phosphoderivatives in biological media (for example, to render phosphorylated enzymes insoluble).
It has been found that alumina can advantageously be used for the purification of phosphopeptides from casein hydrolyzates, producing a mixture of soluble peptides with a high degree of purity, containing a large quantity of phosphoserine.
A first subject of the invention is therefore a method for the selective purification of phosphopeptides from hydrolyzed casein, characterized in that it comprises adsorption of the casein hydrolyzate on alumina and elution of the selectively-adsorbed phosphopeptides with ammonium hydroxide.
Another subject of the invention is a mixture of purified phosphopeptides which can be produced by the above-mentioned selective adsorption and elution method.
The method according to the invention has considerable advantages such as speed, a good purification yield, and an absence of contaminants in the peptides obtained. The use of ammonium hydroxide for the elution of the phosphopeptides constitutes a further improvement since it avoids contaminating the peptides with the phosphorus contained in the buffer and the eluent, and because the eluent can easily be removed in the form of ammonia in the subsequent peptide lyophilization process.
1 ' . . , .
The peptides obtained by the method of the invention are soluble in water and generally have a phosphoserine content equal to at least 25~ of the total amino-acids.
The mixture of phosphopeptides obtained by the method according to the invention can advantageously be used for the preparation of pharmaceutical or dietary compositions for promoting the absorption of metal ions such as calcium, gold, iron, lithium, magnesium and zinc in the human organism; the purified phosphopeptides can also be used for ionography purposes.
The phosphopeptides can also be used in pharmaceutical formulations for anti-caries treatment. The pharmaceutical or dietary compositions preferably comprise the~phosphopeptides in association with lactulose and/or lactose.
The substrate on which the method according to the invention is performed is constituted by a casein hydrolyzate obtainable by enzymatic or chemical hydrolysis. The enzymatic hydrolysis method's are known per se and preferably involve the use of proteolytic enzymes which simulate the protein digestion which takes place in vivo in the human body. For example, it is possible to use pancreatin which is a complex mixture containing trypsin, chymotrypsin and other secondary proteolytic enzymes.
The use of casein hydrolyzates with trypsin or mixtures of oc-chymotrypsin and trypsin is particularly preferred.
The hydrolyzates are obtained by incubation of casein with proteolytic enzymes at pH 8.5 and 37°C (see SIGMA catalogue, 1998). lkg of casein gives approximately 1kg of hydrolyzate from which approximately 150 g of purified phosphopeptides is obtained.
The hydrolyzates produced by chemical methods are obtained by hydrolysis with 1N hydrochloric acid at 100°C under reflux.
' ~ _ ,~ ' , , . , The alumina used in the method according to the invention may be any commercially available activated alumina. During the adsorption on alumina the pH of the hydrolyzed product is preferably regulated to values of between 6.0 and 7.5.
The ammonium hydroxide used is typically an aqueous solution, preferably having an NH3 concentration of from 12 to 15$.
The methods for the preparation of phosphopeptides and their analytical characterization are described in the following examples.
Example 1 - Preparation of phosphopeptides from a tryptic hydrolyzate 125g of tryptic casein hydrolyzate was dissolved in 1.251 of distilled H20, the pH being adjusted to 7.0 at 23°C. 100g of activated alumina was added to the solution and gentle stirring of the suspension was maintained. The pH, which tended to increase, was readjusted to 7.0 with hydrochloric acid.
After 20 minutes, the solid phase was separated by filtration, washed with distilled water and re-suspended in 150m1 of distilled water; the pH was then brought to 9.5 with NH40H, with constant stirring.
After 15 minutes, the mixture was filtered and the alumina was ' washed with 50m1 of distilled H20, the filtrate and washings being collected and then lyophilized. The phosphopeptide fraction was recovered as amorphous powder with a yield of 1.65g and the remaining traces of NH3 were eliminated under vacuum in the presence of concentrated sulphuric acid.
Example 2 - Preparation of phosphopeptides from an acid hydrolyzate Alternatively, 50g of casein was suspended in 500m1 of 1N HC1 in a flask with a reflux condenser with heating to 100°C in a bain .
marie, conditions of incomplete boiling being maintained to prevent dispersion of the solid.
After 150 minutes, the mixture was filtered and the filtrate was brought to pH 6. 50g of neutral activated alumina was added.
After the mixture had been stirred slowly for 30 minutes, the alumina was collected by Buchner, washed with water and re-suspended in 80m1 of water. The pH was brought to 9.5 with concentrated NH40H and the mixture was stirred slowly for one hour.
The liquid phase was collected by filtration, washed with 40m1 of water and lyophilized. The lyophilizate obtained consisted of 2g of phosphopeptides with discrete chromatographic homogeneity, that is, constituted by a mixture of two-three products.
Analytical characterization of the natural phosphopeptides The following tests were carried out with the phosphopeptides obtained from the tryptic hydrolyzate, for the sole reason that, in this case, it is possible to use the starting material, which is soluble, as a control. The characteristics of the products obtained by the two systems, that is, by enzymatic or chemical hydrolysis, are, however, similar.
The phosphopeptides obtained by the methods described above constituted, as stated, a discretely homogeneous material composed mainly of dipeptides containing Glu and P-Ser. To demonstrate this, high-voltage electrophoresis was performed on 20~ polyacrylamide gel.
Tests carried by the conventional Laemmli method as described by Donella-Deana et al (Bioch. Bioph. Res. Comm., 106, 1309, 1989) did not succeed in identifying the phosphopeptides which clearly had molecular weights of less than 1000.
The amino-acid composition of the phosphopeptides was also determined; all contained P-serine and the organic phosphorus determination showed the phosphorus enrichment brought about by the purification method.
Analysis of the amino-acids (as ~ of the total amino-acids) Starting hydrolyzate Phosphopeptide fraction Glu 23.66 25.06 Ser 7.54 25.76 As can be seen, the purified phosphopeptide fraction contained a clearly greater percentage of serine. The content of other amino-acids varied from 1.8 to 6~, approximately.
Organic phosphorus determination The following values were obtained (mean ~ SD of four tests) with the use of Martin and Doty's method (Anal. Chem., 21, 965, 1949) with silicon tungstate as the precipitant and ammonium molybdate as the specific reagent:
Phosphate (~..~.g/mg of casein) Starting hydrolyzate 0.73 ~ 0.05 Phosphopeptide fraction 4.30 ~ 0.35 Interaction with metal salts The chelation capacity for calcium was determined by Ca-specific electrode (Metrohm, calomel reference electrode). The affinity (1/K). of the purified phosphopeptides for calcium was approximately twice that of the hydrolyzate:
Kphosph. 1.7 mg/ml Khydrol. 3. 6 mg/ml.
P w The chelation capacity for iron and gold ions was investigated in solution by a conductometric method, by means of a Radiometer CDM3 conductivity meter.
Solutions of FeCl3 and of AuCl4H in double-distilled H20 were used at concentrations of from 0.2 to 1.4 mM and the phosphopeptides were used at concentrations of from 0.5 to 250 mg/ml.
The relative affinities of the purified phosphopeptides for Fe and Au were, respectively:
Fe 1.1 mg/ml Au 1.2 mg/ml.
Similar chelation properties were found for zinc. The results obtained are summarized in Tables 1 and 2.
Results obtained with the Ca-specific electrode, expressed as percentage of Ca chelated in dependence on increasing concentrations of starting tryptic peptones or of purified phosphopeptides Concentration (mg/ml) Tryptic peptones Phosphopeptides 15.0 98 100 12.5 90 98 10.0 88 94 7.5 74 88 5.0 62 82 2.5 40 63 Values of the dissociation constants for the following equilibria can be obtained from this data by a graphical method:
Peptones + Ca ---> peptones-Ca r i , . . .
__ phosphopeptides + Ca ---> Phosphopeptides-Ca Results of chelation tests performed by a conductometric method by mixing increasing quantities of (Ca, Fe, Au) ions with a fixed quantity of phosphopeptides (250 mg/ml) in a final volume of 50 ml of double-distilled water.
The quantity of ions added (in ~.1 of 1 M solutions) can be compared with the conductivity values, expressed in ~s.
Subtraction of the conductivity relating to the phosphopeptides alone (F) from that relating to the phosphopeptide-ion mixture gives values which differ to a greater extent from those relating the ions alone the greater is the degree of chelation.
Quantity of Ca Conductivity Conductivity Conductivity Ca (F+Ca) - F (F+Ca) 0.0000 1.2000 0.0000 100.00 10.000 38.000 20.000 120.00 20.000 73.000 50.000 150.00 30.000 115.00 90.000 190.00 40.000 150.00 125.00 225.00 50.000 190.00 150.00 250.00 Quantity of Ca Conductivity Conductivity Conductivity Ca (F+Ca) - F (F+Ca) 60.000 225.00 185.00 285.00 70.000 260.00 210.00 310.00 Quantity ConductivityConductivityConductivi~y o Fo Fe (FTFe) - (FTFe) F
0.0000 1.2000 0.0000 100.00 10.000 90.000 0.0000 90.000 20.000 200.00 50.000 150.00 30.000 260.00 150.00 250.00 40_000 360.00 250.00 350.00 50.000 450.00 350.00 450.00 60.000 500.00 400.00 500.00 70.000 600.00 500.00 600.00 Quantity ConductivityConductivityConductivity of Au Au (F+Au) - (F+Au) F
0.0000 1.2000 0.0000 100.00 10.000 80.000 0.0000 90.000 20.000 210.00 15.000 115.00 30.000 300.00 100.00 200.00 40.000 410.00 190.00 290.00 50.000 470.00 270.00 370.00 60.000 550.00 325.00 425.00 70_000 610.00 420.00 520.00 To demonstrate that the chelation capacity of the phosphopeptides of the invention is not an aspecific effect, a comparison test was carried ouL with the use of non-phosphorylated peptides such as glycyl glycine and enkephaline.
In the case of enkephaline and Qlycyl Qlycine, the values obtained as the difference of (peptide + ion) - peptide coincide with those relating to the conductivity of the ion alone.
This means that no chelation of Ca, of Fe, or of Au was evident in the conditions used.
Another subject of the invention is therefore constituted by the use of the peptides which can be produced by the above-described method to facilitate intestinal absorption of metal ions such as calcium, iron, lithium, magnesium, gold and zinc.
Effect of phosphopeptides on the uptake of 45Ca "per os" in the rat Twelve male Wistar rats (350g body weight) were divided into three groups and housed for three days in cages for two with food "ad libitum". A quantity of 16.6~,Ci of 45Ca (+ 1mM Ca cold;
approximately 7x106 cpm) was introduced into the bottles containing the drinking water and the quantity of water drunk in the course of three days (about 300 ml) was measured at the end of the test, when the rats were killed by decapitation. The whole blood (5 ml), heparinized, was deproteinized with 10~
final trichloroacetic acid and the residual radioactivity was determined by scintillation.
The radioactivity values (mean ~ SD of four determinations) were as follows:
Groups Radioactivity (cpm/rat) Control 683 ~ 68 +,.1 g hydrolyzate/500 ml 640 ~ 55 + 1 g phosphopeptides/500 ml 2240 ~ 98 As can be seen, the amount of calcium absorbed by the rats was increased more than three times by the phosphopeptides and remained unchanged by the hydrolyzate.
Effect of phosphopeptides on intestinal absorption of calcium in man < i . s a Six healthy volunteers ingested lg of calcium gluconate (Sandoz) and their urine was collected for 24 hours. After a "wash out"
period of 48 hours, they ingested lg of calcium plus 2.8g of phosphopeptides dissolved in 200m1 of water and their urine was collected for 24 hours.
Urinary excretion of calcium was then evaluated as shown in Table 3.
Urinary excretion of calcium in man (m = male; f = female) Subjects Calcium (mmo1/die)Calcium+phosphopeptides'(mmol/die) L.G. m 7.50 8.50 colorimet. met.
(58) (arsenazo II) M.B. f 1.51 1.40 (56) A.L. m 7.48 9.43 (45) G.M. f 7.14 mean=5.69 6.95 mean=6.4 (41) S.B. f 3.71 4.92 (35) M.G. m 6.80 7.20 (51) The peptide L-Glu-L-Ser-P was synthesized chemically and it was found that the effects of the natural peptides were reproduced faithfully by the synthetic peptide.
The mixture of phosphopeptides obtained by the method according to the invention can advantageously be used for the preparation of pharmaceutical or dietary compositions for promoting the absorption of metal ions such as calcium, gold, iron, lithium, magnesium and zinc in the human organism; the purified phosphopeptides can also be used for ionography purposes.
The phosphopeptides can also be used in pharmaceutical formulations for anti-caries treatment. The pharmaceutical or dietary compositions preferably comprise the~phosphopeptides in association with lactulose and/or lactose.
The substrate on which the method according to the invention is performed is constituted by a casein hydrolyzate obtainable by enzymatic or chemical hydrolysis. The enzymatic hydrolysis method's are known per se and preferably involve the use of proteolytic enzymes which simulate the protein digestion which takes place in vivo in the human body. For example, it is possible to use pancreatin which is a complex mixture containing trypsin, chymotrypsin and other secondary proteolytic enzymes.
The use of casein hydrolyzates with trypsin or mixtures of oc-chymotrypsin and trypsin is particularly preferred.
The hydrolyzates are obtained by incubation of casein with proteolytic enzymes at pH 8.5 and 37°C (see SIGMA catalogue, 1998). lkg of casein gives approximately 1kg of hydrolyzate from which approximately 150 g of purified phosphopeptides is obtained.
The hydrolyzates produced by chemical methods are obtained by hydrolysis with 1N hydrochloric acid at 100°C under reflux.
' ~ _ ,~ ' , , . , The alumina used in the method according to the invention may be any commercially available activated alumina. During the adsorption on alumina the pH of the hydrolyzed product is preferably regulated to values of between 6.0 and 7.5.
The ammonium hydroxide used is typically an aqueous solution, preferably having an NH3 concentration of from 12 to 15$.
The methods for the preparation of phosphopeptides and their analytical characterization are described in the following examples.
Example 1 - Preparation of phosphopeptides from a tryptic hydrolyzate 125g of tryptic casein hydrolyzate was dissolved in 1.251 of distilled H20, the pH being adjusted to 7.0 at 23°C. 100g of activated alumina was added to the solution and gentle stirring of the suspension was maintained. The pH, which tended to increase, was readjusted to 7.0 with hydrochloric acid.
After 20 minutes, the solid phase was separated by filtration, washed with distilled water and re-suspended in 150m1 of distilled water; the pH was then brought to 9.5 with NH40H, with constant stirring.
After 15 minutes, the mixture was filtered and the alumina was ' washed with 50m1 of distilled H20, the filtrate and washings being collected and then lyophilized. The phosphopeptide fraction was recovered as amorphous powder with a yield of 1.65g and the remaining traces of NH3 were eliminated under vacuum in the presence of concentrated sulphuric acid.
Example 2 - Preparation of phosphopeptides from an acid hydrolyzate Alternatively, 50g of casein was suspended in 500m1 of 1N HC1 in a flask with a reflux condenser with heating to 100°C in a bain .
marie, conditions of incomplete boiling being maintained to prevent dispersion of the solid.
After 150 minutes, the mixture was filtered and the filtrate was brought to pH 6. 50g of neutral activated alumina was added.
After the mixture had been stirred slowly for 30 minutes, the alumina was collected by Buchner, washed with water and re-suspended in 80m1 of water. The pH was brought to 9.5 with concentrated NH40H and the mixture was stirred slowly for one hour.
The liquid phase was collected by filtration, washed with 40m1 of water and lyophilized. The lyophilizate obtained consisted of 2g of phosphopeptides with discrete chromatographic homogeneity, that is, constituted by a mixture of two-three products.
Analytical characterization of the natural phosphopeptides The following tests were carried out with the phosphopeptides obtained from the tryptic hydrolyzate, for the sole reason that, in this case, it is possible to use the starting material, which is soluble, as a control. The characteristics of the products obtained by the two systems, that is, by enzymatic or chemical hydrolysis, are, however, similar.
The phosphopeptides obtained by the methods described above constituted, as stated, a discretely homogeneous material composed mainly of dipeptides containing Glu and P-Ser. To demonstrate this, high-voltage electrophoresis was performed on 20~ polyacrylamide gel.
Tests carried by the conventional Laemmli method as described by Donella-Deana et al (Bioch. Bioph. Res. Comm., 106, 1309, 1989) did not succeed in identifying the phosphopeptides which clearly had molecular weights of less than 1000.
The amino-acid composition of the phosphopeptides was also determined; all contained P-serine and the organic phosphorus determination showed the phosphorus enrichment brought about by the purification method.
Analysis of the amino-acids (as ~ of the total amino-acids) Starting hydrolyzate Phosphopeptide fraction Glu 23.66 25.06 Ser 7.54 25.76 As can be seen, the purified phosphopeptide fraction contained a clearly greater percentage of serine. The content of other amino-acids varied from 1.8 to 6~, approximately.
Organic phosphorus determination The following values were obtained (mean ~ SD of four tests) with the use of Martin and Doty's method (Anal. Chem., 21, 965, 1949) with silicon tungstate as the precipitant and ammonium molybdate as the specific reagent:
Phosphate (~..~.g/mg of casein) Starting hydrolyzate 0.73 ~ 0.05 Phosphopeptide fraction 4.30 ~ 0.35 Interaction with metal salts The chelation capacity for calcium was determined by Ca-specific electrode (Metrohm, calomel reference electrode). The affinity (1/K). of the purified phosphopeptides for calcium was approximately twice that of the hydrolyzate:
Kphosph. 1.7 mg/ml Khydrol. 3. 6 mg/ml.
P w The chelation capacity for iron and gold ions was investigated in solution by a conductometric method, by means of a Radiometer CDM3 conductivity meter.
Solutions of FeCl3 and of AuCl4H in double-distilled H20 were used at concentrations of from 0.2 to 1.4 mM and the phosphopeptides were used at concentrations of from 0.5 to 250 mg/ml.
The relative affinities of the purified phosphopeptides for Fe and Au were, respectively:
Fe 1.1 mg/ml Au 1.2 mg/ml.
Similar chelation properties were found for zinc. The results obtained are summarized in Tables 1 and 2.
Results obtained with the Ca-specific electrode, expressed as percentage of Ca chelated in dependence on increasing concentrations of starting tryptic peptones or of purified phosphopeptides Concentration (mg/ml) Tryptic peptones Phosphopeptides 15.0 98 100 12.5 90 98 10.0 88 94 7.5 74 88 5.0 62 82 2.5 40 63 Values of the dissociation constants for the following equilibria can be obtained from this data by a graphical method:
Peptones + Ca ---> peptones-Ca r i , . . .
__ phosphopeptides + Ca ---> Phosphopeptides-Ca Results of chelation tests performed by a conductometric method by mixing increasing quantities of (Ca, Fe, Au) ions with a fixed quantity of phosphopeptides (250 mg/ml) in a final volume of 50 ml of double-distilled water.
The quantity of ions added (in ~.1 of 1 M solutions) can be compared with the conductivity values, expressed in ~s.
Subtraction of the conductivity relating to the phosphopeptides alone (F) from that relating to the phosphopeptide-ion mixture gives values which differ to a greater extent from those relating the ions alone the greater is the degree of chelation.
Quantity of Ca Conductivity Conductivity Conductivity Ca (F+Ca) - F (F+Ca) 0.0000 1.2000 0.0000 100.00 10.000 38.000 20.000 120.00 20.000 73.000 50.000 150.00 30.000 115.00 90.000 190.00 40.000 150.00 125.00 225.00 50.000 190.00 150.00 250.00 Quantity of Ca Conductivity Conductivity Conductivity Ca (F+Ca) - F (F+Ca) 60.000 225.00 185.00 285.00 70.000 260.00 210.00 310.00 Quantity ConductivityConductivityConductivi~y o Fo Fe (FTFe) - (FTFe) F
0.0000 1.2000 0.0000 100.00 10.000 90.000 0.0000 90.000 20.000 200.00 50.000 150.00 30.000 260.00 150.00 250.00 40_000 360.00 250.00 350.00 50.000 450.00 350.00 450.00 60.000 500.00 400.00 500.00 70.000 600.00 500.00 600.00 Quantity ConductivityConductivityConductivity of Au Au (F+Au) - (F+Au) F
0.0000 1.2000 0.0000 100.00 10.000 80.000 0.0000 90.000 20.000 210.00 15.000 115.00 30.000 300.00 100.00 200.00 40.000 410.00 190.00 290.00 50.000 470.00 270.00 370.00 60.000 550.00 325.00 425.00 70_000 610.00 420.00 520.00 To demonstrate that the chelation capacity of the phosphopeptides of the invention is not an aspecific effect, a comparison test was carried ouL with the use of non-phosphorylated peptides such as glycyl glycine and enkephaline.
In the case of enkephaline and Qlycyl Qlycine, the values obtained as the difference of (peptide + ion) - peptide coincide with those relating to the conductivity of the ion alone.
This means that no chelation of Ca, of Fe, or of Au was evident in the conditions used.
Another subject of the invention is therefore constituted by the use of the peptides which can be produced by the above-described method to facilitate intestinal absorption of metal ions such as calcium, iron, lithium, magnesium, gold and zinc.
Effect of phosphopeptides on the uptake of 45Ca "per os" in the rat Twelve male Wistar rats (350g body weight) were divided into three groups and housed for three days in cages for two with food "ad libitum". A quantity of 16.6~,Ci of 45Ca (+ 1mM Ca cold;
approximately 7x106 cpm) was introduced into the bottles containing the drinking water and the quantity of water drunk in the course of three days (about 300 ml) was measured at the end of the test, when the rats were killed by decapitation. The whole blood (5 ml), heparinized, was deproteinized with 10~
final trichloroacetic acid and the residual radioactivity was determined by scintillation.
The radioactivity values (mean ~ SD of four determinations) were as follows:
Groups Radioactivity (cpm/rat) Control 683 ~ 68 +,.1 g hydrolyzate/500 ml 640 ~ 55 + 1 g phosphopeptides/500 ml 2240 ~ 98 As can be seen, the amount of calcium absorbed by the rats was increased more than three times by the phosphopeptides and remained unchanged by the hydrolyzate.
Effect of phosphopeptides on intestinal absorption of calcium in man < i . s a Six healthy volunteers ingested lg of calcium gluconate (Sandoz) and their urine was collected for 24 hours. After a "wash out"
period of 48 hours, they ingested lg of calcium plus 2.8g of phosphopeptides dissolved in 200m1 of water and their urine was collected for 24 hours.
Urinary excretion of calcium was then evaluated as shown in Table 3.
Urinary excretion of calcium in man (m = male; f = female) Subjects Calcium (mmo1/die)Calcium+phosphopeptides'(mmol/die) L.G. m 7.50 8.50 colorimet. met.
(58) (arsenazo II) M.B. f 1.51 1.40 (56) A.L. m 7.48 9.43 (45) G.M. f 7.14 mean=5.69 6.95 mean=6.4 (41) S.B. f 3.71 4.92 (35) M.G. m 6.80 7.20 (51) The peptide L-Glu-L-Ser-P was synthesized chemically and it was found that the effects of the natural peptides were reproduced faithfully by the synthetic peptide.
Claims (8)
1. A method for the selective purification of phosphopeptides from hydrolyzed casein, characterized in that it comprises adsorption of the casein hydrolyzate on alumina and elution of the phosphopeptides selectively adsorbed on alumina with ammonium hydroxide.
2. A method according to Claim 1, in which the casein hydrolyzate is a hydrolyzate produced by a chemical or an enzymatic method.
3. A method according to Claim 2, in which the hydrolyzate produced by an enzymatic method is a tryptic hydrolyzate.
4. A purified mixture of phosphopeptides as obtainable by a method of adsorption of casein hydrolyzate on alumina and elution with ammonium hydroxide.
5. A mixture of phosphopeptides according to Claim 4, having a phosphoserine content equal to at least 25% of the total aminoacids.
6. A mixture of phosphopeptides according to Claim 4 or Claim 5, containing phosphopeptides soluble in water and substantially free of non-phosphorylated peptides.
7. A pharmaceutical composition comprising a mixture of phosphopeptides according to any one of Claims 4 to 6 for use as a medicament for promoting intestinal absorption of calcium, gold, iron, lithium, magnesium or zinc ions.
8. Use of a mixture of phosphopeptides according to any one of Claims 4 to 6 in ionography.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ITT098A000891 | 1998-10-21 | ||
| ITTO980891 IT1305125B1 (en) | 1998-10-21 | 1998-10-21 | Selective purification of phosphopeptides from hydrolyzed casein for promoting intestinal absorption of essential ions and in ionography comprises adsorbing casein hydrolyzate on alumina and eluting phosphopeptides |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2286971A1 true CA2286971A1 (en) | 2000-04-21 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2286971 Abandoned CA2286971A1 (en) | 1998-10-21 | 1999-10-20 | A method for the purification of natural phosphorylated peptides, a mixture of phosphopeptides thus obtained and the use thereof |
Country Status (2)
| Country | Link |
|---|---|
| CA (1) | CA2286971A1 (en) |
| IT (1) | IT1305125B1 (en) |
-
1998
- 1998-10-21 IT ITTO980891 patent/IT1305125B1/en active
-
1999
- 1999-10-20 CA CA 2286971 patent/CA2286971A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| ITTO980891A1 (en) | 2000-04-21 |
| IT1305125B1 (en) | 2001-04-10 |
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