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Peptides bound to albumin
Life Sciences, Vol. 39, pp. 1751-1753 Printed in the U.S.A.
Pergamon Journals
PEPTIDES BOUND TO ALBUMIN Yves MENEZO and Chaqu~ KHATCHADOURIAN INRA, INSA, Biologie 406, 69621VILLEURBANNE
Cedex, FRANCE
(Received in final form August 8, 1986)
SUMMARY Preparations of albumin (placenta or serum) do bind peptides. Dissociation of these peptides from the albumin core can occur partially in water but is much more efficient in the presence of amino acids and salts, in complex culture media for example. Contrary to common belief, we observed that arginin vasopressin (AVP) can bind to serum albumin, at least in commercial powder form. INTRODUCTION Albumins from various origin (serum, placenta) are commonly added to culture media (I, 2, 3) to replace serum ; they are also used in biological buffers for protein (enzymes, hormones) isolation and preparation. This macromolecule is necessary for the "in vitro" growth of cell lines. For embryos, even if it is not a strict requirement (4), albumin may be essential, in the absence of serum, for particular stages of embryo developpement. The true and complete role of albumin remains largely unknown, even though it is a carrier of lipids and steroid hormones. Some data indicate that albumin could bind growth factors of low molecular weight. (5) In this experiment, we have defined the ability of peptides (of various molecular weight) to bind to corm~ercial preparations of albumin, including hormones not usually considered as albumin ligands. MATERIAL AND METHODS Peptides bound to albumin Analysis were performed on 3 batches of bovine serum albumin, (bSA SIGMA A 4378, batches No 94 F 9405, 14 F 9345, 125 F 9350) and on human placental albumin, (hPA MERIEUX), which was checked for its purity by immunoelectrophoresis and high performance liquid chromatography (3). Both types of albumin were dissolved either in a complete culture medium mixture (3), or in water to a 1% final concentration. The peptide amino acids bound to albumin were determined according to the following scheme : A) Ultrafiltration was performed on UM IO membranes (Amicon) B) Gel filtration was performed on Sephadex G25 (Elution System Acetic Acid 5%) C) Gel filtration was performed on Sephadex GI5 with the same Elution system HPLC : Protein Pak Waters E 125, with Phosphate buffer pH 7.0, 0.05 M as an eluant. D) Hydrolysis was performed in 6N HCI, 12 hours at IIO°C, with thioglycolic acid (0.5%) as a reducin~ agent. E) Amino acid analysis was performed on the amino acid analyzer Kontron Liquimat III. Copyright
0024-3205/86 $3.00 + .00 (c) 1986 Pergamon Journals Ltd.
1752
Peptides Bound to Albumin
Vol. 39, No. 19, 1986
Albumin (bSA or hPA)
Dissolved in Water
Dissolved in a complex culture medium
A) ULTRAFILTRATION NN • 10000
A) ULTRAFILTRATION NW < 10000
B) Sephalex G25| (180 , MW < 2000 collected)
C) Sephadex GI5 or HPLC
D) Hydrolysis 12h 6N HCI - IIO°C
E) Amino acid Analysis
\
J I
Amino acid Analysis
D) Hydrolysis 12h 6N HCL - IIO°C
Amino acid Analysis
/
\
Peptide A.A. bound to Albumin
Amino acid Analysis
/
Peptide A.A. bound to Albumin
Determination of arginin vasopress~n It was carried out by the dissolution of bovine serum albumin (from 6 different batches : 94 F 9405, 94 F 9505, 94 F 9410, 94 F 9415, 14 F 9345, 125 F 9350) in complex culture medium, at a 1% final level. The solution was then filtered and stored during 15 days at 4=C. Then, the samples were analysed and quantitative estimates were made according toRIA. (6). RESULTS Peptides bound to Albumin The data obtained by HPLC and Sephaxex GI5, which are similar are presente~ in Table I. Peptides released from albumin in a complex medium account for 1 to 2% of the total albumin weight. However dissociation from albumin is less important in water (~O.1%). Direct analysis of ultrafiltration in water never offered detectable amounts of free amino acids. Determination of arginin vasopressin For the six batches, the average amount of AVP released by dissociation from the bovine serum albumin (in complex medium) was 306 ! 50 pg per gram of bovine serum albumin. DISCUSSION ~ a t e v e r its source, albumin is bound effectively by peptides. If we consider the level of dissociation in water and in a complex culture medium, then it really seems that dissociation is related to chemical factors, and peptide dissociation is due to competition with the free a~ino acids. If we consider AVP "per se", then it is probable that the hormone co-precipitates during prepara-
Vol. 39, No. 19, 1986
Peptides Bound to Albumin
1753
Table I. Peptides released from Albumin : Percentage of each amino acid (+ Amounts detected but not quantified) Complete medium
Aminoacid
bSA
Asp + AsN Threo Ser GI~ + G l n Pro Gly Ala Val Cyst Met lleu Leu Tyr Phe Lys Hist Arg OHLy s
Water
hPA
bSA
hPA
4.0 4.3 0.7 21.2 0.8 54.8 + 3.7 + + 0.4 1.3 0.3 2.5 3.4 0.9 1.7 +
8.3 4.0 I.I 28.6 + 45.1 I .O 5.0 + + 0.3 0.7 + 1.0 2.8 0.6 1.5 +
7.7 4.7 5.6 16.9 2.1 5.8 6.8 6.9 + I .6 2.4 12.5 + 6.3 6.6 3.8 8.7 1.7
9.9 4.6 4.0 11.7 2.3 7.8 6.3 4.5 10.3 i .8 3.5 6.9 3.7 7.O 5.5 3.1 4.4 2.6
Total (mg. g-l) 19.1 release
15.7
0.66
0.49
tion. Then in solution a new equilibrium occurs between the bound and free hormone. In any case, one must take care when using albumin powders with either "in vivo" or in "in vitro" systems, of these equilibrium variations. Of equal importance, bSA has been claimed to be, in some "in vitro" experiments, the sole nitrogen source of the embryo ; it is probable that the true nitrogen source, available for further synthesis, is the low MW peptides and not the macromolecule "per se." In conclusion, albumin is a mostly undefined molecule that is able, in its powder form, to carry unexpected components,dissociated after solubilization in complex media. The bound llgands may vary according to the physiological state of the animals at the time of serum collection. ACKNOWLEDGEMENT S We thank Dr. ARDAILLOU for the determination
of AVP.
REFERENCES i - BRINSTER R.L., J. Exp. Zool. 158. 69-78 (1965) 2 - BRACKETT (B.G.) in "Fertilization and embryonic development in vitro" L.J. MASTROIANNI and J.D. BIGGERS ed., Plenum Press (1981). 3 - M E N E Z O (Y.), TESTART (J.), PEDRONE (D.), Fertil. Steril. 42, 750-755 (1984) 4 - CARO (C.M.), TROUNSON (A.), J. Vitro fert. Embryo transfer, i, 183-187 (1984) 5 - K A N E (M.T.), J. Reprod. fert. 73, 147-150 (1985) 6 - ROBERTSON (G.L.), MAHR (E.A.), ATHAR (S.), SINMA (T.), J. Clin. Invest. 52, 5340-5345 (1973).
Pergamon Journals
PEPTIDES BOUND TO ALBUMIN Yves MENEZO and Chaqu~ KHATCHADOURIAN INRA, INSA, Biologie 406, 69621VILLEURBANNE
Cedex, FRANCE
(Received in final form August 8, 1986)
SUMMARY Preparations of albumin (placenta or serum) do bind peptides. Dissociation of these peptides from the albumin core can occur partially in water but is much more efficient in the presence of amino acids and salts, in complex culture media for example. Contrary to common belief, we observed that arginin vasopressin (AVP) can bind to serum albumin, at least in commercial powder form. INTRODUCTION Albumins from various origin (serum, placenta) are commonly added to culture media (I, 2, 3) to replace serum ; they are also used in biological buffers for protein (enzymes, hormones) isolation and preparation. This macromolecule is necessary for the "in vitro" growth of cell lines. For embryos, even if it is not a strict requirement (4), albumin may be essential, in the absence of serum, for particular stages of embryo developpement. The true and complete role of albumin remains largely unknown, even though it is a carrier of lipids and steroid hormones. Some data indicate that albumin could bind growth factors of low molecular weight. (5) In this experiment, we have defined the ability of peptides (of various molecular weight) to bind to corm~ercial preparations of albumin, including hormones not usually considered as albumin ligands. MATERIAL AND METHODS Peptides bound to albumin Analysis were performed on 3 batches of bovine serum albumin, (bSA SIGMA A 4378, batches No 94 F 9405, 14 F 9345, 125 F 9350) and on human placental albumin, (hPA MERIEUX), which was checked for its purity by immunoelectrophoresis and high performance liquid chromatography (3). Both types of albumin were dissolved either in a complete culture medium mixture (3), or in water to a 1% final concentration. The peptide amino acids bound to albumin were determined according to the following scheme : A) Ultrafiltration was performed on UM IO membranes (Amicon) B) Gel filtration was performed on Sephadex G25 (Elution System Acetic Acid 5%) C) Gel filtration was performed on Sephadex GI5 with the same Elution system HPLC : Protein Pak Waters E 125, with Phosphate buffer pH 7.0, 0.05 M as an eluant. D) Hydrolysis was performed in 6N HCI, 12 hours at IIO°C, with thioglycolic acid (0.5%) as a reducin~ agent. E) Amino acid analysis was performed on the amino acid analyzer Kontron Liquimat III. Copyright
0024-3205/86 $3.00 + .00 (c) 1986 Pergamon Journals Ltd.
1752
Peptides Bound to Albumin
Vol. 39, No. 19, 1986
Albumin (bSA or hPA)
Dissolved in Water
Dissolved in a complex culture medium
A) ULTRAFILTRATION NN • 10000
A) ULTRAFILTRATION NW < 10000
B) Sephalex G25| (180 , MW < 2000 collected)
C) Sephadex GI5 or HPLC
D) Hydrolysis 12h 6N HCI - IIO°C
E) Amino acid Analysis
\
J I
Amino acid Analysis
D) Hydrolysis 12h 6N HCL - IIO°C
Amino acid Analysis
/
\
Peptide A.A. bound to Albumin
Amino acid Analysis
/
Peptide A.A. bound to Albumin
Determination of arginin vasopress~n It was carried out by the dissolution of bovine serum albumin (from 6 different batches : 94 F 9405, 94 F 9505, 94 F 9410, 94 F 9415, 14 F 9345, 125 F 9350) in complex culture medium, at a 1% final level. The solution was then filtered and stored during 15 days at 4=C. Then, the samples were analysed and quantitative estimates were made according toRIA. (6). RESULTS Peptides bound to Albumin The data obtained by HPLC and Sephaxex GI5, which are similar are presente~ in Table I. Peptides released from albumin in a complex medium account for 1 to 2% of the total albumin weight. However dissociation from albumin is less important in water (~O.1%). Direct analysis of ultrafiltration in water never offered detectable amounts of free amino acids. Determination of arginin vasopressin For the six batches, the average amount of AVP released by dissociation from the bovine serum albumin (in complex medium) was 306 ! 50 pg per gram of bovine serum albumin. DISCUSSION ~ a t e v e r its source, albumin is bound effectively by peptides. If we consider the level of dissociation in water and in a complex culture medium, then it really seems that dissociation is related to chemical factors, and peptide dissociation is due to competition with the free a~ino acids. If we consider AVP "per se", then it is probable that the hormone co-precipitates during prepara-
Vol. 39, No. 19, 1986
Peptides Bound to Albumin
1753
Table I. Peptides released from Albumin : Percentage of each amino acid (+ Amounts detected but not quantified) Complete medium
Aminoacid
bSA
Asp + AsN Threo Ser GI~ + G l n Pro Gly Ala Val Cyst Met lleu Leu Tyr Phe Lys Hist Arg OHLy s
Water
hPA
bSA
hPA
4.0 4.3 0.7 21.2 0.8 54.8 + 3.7 + + 0.4 1.3 0.3 2.5 3.4 0.9 1.7 +
8.3 4.0 I.I 28.6 + 45.1 I .O 5.0 + + 0.3 0.7 + 1.0 2.8 0.6 1.5 +
7.7 4.7 5.6 16.9 2.1 5.8 6.8 6.9 + I .6 2.4 12.5 + 6.3 6.6 3.8 8.7 1.7
9.9 4.6 4.0 11.7 2.3 7.8 6.3 4.5 10.3 i .8 3.5 6.9 3.7 7.O 5.5 3.1 4.4 2.6
Total (mg. g-l) 19.1 release
15.7
0.66
0.49
tion. Then in solution a new equilibrium occurs between the bound and free hormone. In any case, one must take care when using albumin powders with either "in vivo" or in "in vitro" systems, of these equilibrium variations. Of equal importance, bSA has been claimed to be, in some "in vitro" experiments, the sole nitrogen source of the embryo ; it is probable that the true nitrogen source, available for further synthesis, is the low MW peptides and not the macromolecule "per se." In conclusion, albumin is a mostly undefined molecule that is able, in its powder form, to carry unexpected components,dissociated after solubilization in complex media. The bound llgands may vary according to the physiological state of the animals at the time of serum collection. ACKNOWLEDGEMENT S We thank Dr. ARDAILLOU for the determination
of AVP.
REFERENCES i - BRINSTER R.L., J. Exp. Zool. 158. 69-78 (1965) 2 - BRACKETT (B.G.) in "Fertilization and embryonic development in vitro" L.J. MASTROIANNI and J.D. BIGGERS ed., Plenum Press (1981). 3 - M E N E Z O (Y.), TESTART (J.), PEDRONE (D.), Fertil. Steril. 42, 750-755 (1984) 4 - CARO (C.M.), TROUNSON (A.), J. Vitro fert. Embryo transfer, i, 183-187 (1984) 5 - K A N E (M.T.), J. Reprod. fert. 73, 147-150 (1985) 6 - ROBERTSON (G.L.), MAHR (E.A.), ATHAR (S.), SINMA (T.), J. Clin. Invest. 52, 5340-5345 (1973).