A Potentiometric and Spectroscopic Study of the Proton, and Copper I III and Zinc (III Complexes Formed by Fibrinopeptide A Jean-Francois
Galey, Henryk Kozlowski,
and Leslie D. Pettit
J-FG. Laboratoire
de Chimie Macromoleculaire, Universitk des Sciences et Techniques de Lille, France.-HK. Institute of Chemistry, University of Wroclaw, Poland.-LDP. Department of Inorganic Chemistry, The University, Leeds, U.K.
Results are reported for a potentiometric and spectroscopic (visible, CD, and EPR) study of the complexes of fibrinopeptide A (Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg) with H+, Cu’+, and Zn’+. They show that the peptide forms stable complexes with Cu’+, largely as a result of the Asp2 residue, and that its coordination behavior is almost identical to that Iof the N-terminal tetrapeptide fragment, Ala-Asp-Ser-Gly. Hence the influence of the remaining amino acid residues on coordination to Cu2+ is insignificant.
INTRODUCTION Human
fibrinopeptide
A is a hexadecapeptide,
Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg, by thrombin when there is some
Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-
which is released from fibrinogen on cleavage injury to the body. The local concentration of the
peptide near the site of an injury is high and it contains amino acid residues which are able to coordinate strongly to metal ions. An earlier study of the N-terminal tetrapeptide fragment of fibrinopeptide A, Ala-Asp-Ser-Gly, demonstrated very effective coordination of N-terminal ‘residues to Cu(I1) as a result of the @carboxylate group of the Asp residue in the second position [l]. This fragment could model only the metal binding power of the N-terminal residues and so assumes the absence of steric constraints from the spatial organization of the hexadecapeptide and of competitive coordination to amino acid residues further along the chain. These assumptions appear reasonable but require confirmation by a study of the naturally occurring ligand. We therefore report the synthesis of fibrinopeptide A and the
Address correspondence Leeds LS2 9JT, U.K. Journal of Inorganic 0
to: Dr. Leslie D. Pettit, Department
Biochemistry,
1991 Elsevier Science Publishing
of Inorganic
Chemistry,
The University,
149
44, 149- 153 (1991)
Co., Inc., 655 Avenue of the Americas,
NY. NY 10010
0162-0134/91/$3.50
150
J-F. Galey et al.
results Cu(II)
of potentiometric ZniII).
and spectroscopic
studies
of the complexc~
formed
with
and
EXPERIMENTAL Peptide
Synthesis Fihrinopeptide A was synthesised using Merrifield solid phase synthetic methods based on 1% crosslinked chloromethylated resin (Novabiochem! 121. The staring material, resin-Boc-Arg-Tos (Tos -=-roluene-4-suphonatc). ~a’\ preparcd from the resin and Boc-At-g-Tos b> the cebium salt method [?I; amino ac& ilfloq~rotectcd~ were obtained commcrciallq iNovabiochem. Propeptide (II’ Bachcn,i Rcacti\ t‘ \itlt: chains were protected as follows: Arg by the To> group: llsp’ :irld (;i\?’ ’ 11~the cyclohexyl group because these residues follow Ala and protection uGrt3 cyclohe\~l minimizes /S--migration: Asp-‘, GILI'. and Ser by the henqi group. i‘icavagc ~ti peptide from the resin and cieavage of protecting b‘croup\ from Ihi: pilptide i\r"rC performed using HF in the prcsencc of paracrrso!. T’hc r1:huiting hcu&,apepridc was purified by gel filtration i-I?X gel) and purity was chci,i;c*il b>’ ~hr-l~Il~aiogl-;tl~~~ (paper and HPLC) and b> potentiometry. Amino ai.ici ana1y \ I* liar ,r~crf~rrrm.xl‘IiTCI peptide hydrolysis with h mol dm ’ HC’1 using a Beckman ‘W(i ;!r~~l\~/~r and i:atc the results: Asp, 1.Ol: Ser. 0.81: Glu. 2.06: Gly. 5.01. ,Aia. J.01 1 V) cpcctra 0:3 .I Mvlarh III Jobin-Yvon dichrograph ill the 220--800 nm region. .411 CD rc~llt~ arc c\-pn’+cd in terms of 14~ =:-f1 -- tr. Electron paramagnetic resonance CEPR) spectra were recorded ton a I‘arian El02 spectrometer at liquid nitrogen temperature at 9. 13 ciIJ/, Potentiometric
Studies
Stability constants for H ’ . Cu’ ’ . and Zn’ ’ complexes were calculated from titration curves carried out at 25 “C using total volumes of 1.S cm ‘. Alkali was added from a 0. I cm’ micrometer syringe which had been calibrated by both weight [Itration ;md the titration of standardized materials. Experimental details were: peptide concentration. 0.0025 mnl dm ‘; copper concentration, 0.001 -0.002 tnol dm ‘: ionic strength. 0. 10 mvl dm ’ (KNO,): pH range for complexation,
Galey, Henryk Kozlowski,
and Leslie D. Pettit
J-FG. Laboratoire
de Chimie Macromoleculaire, Universitk des Sciences et Techniques de Lille, France.-HK. Institute of Chemistry, University of Wroclaw, Poland.-LDP. Department of Inorganic Chemistry, The University, Leeds, U.K.
Results are reported for a potentiometric and spectroscopic (visible, CD, and EPR) study of the complexes of fibrinopeptide A (Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg) with H+, Cu’+, and Zn’+. They show that the peptide forms stable complexes with Cu’+, largely as a result of the Asp2 residue, and that its coordination behavior is almost identical to that Iof the N-terminal tetrapeptide fragment, Ala-Asp-Ser-Gly. Hence the influence of the remaining amino acid residues on coordination to Cu2+ is insignificant.
INTRODUCTION Human
fibrinopeptide
A is a hexadecapeptide,
Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg, by thrombin when there is some
Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-
which is released from fibrinogen on cleavage injury to the body. The local concentration of the
peptide near the site of an injury is high and it contains amino acid residues which are able to coordinate strongly to metal ions. An earlier study of the N-terminal tetrapeptide fragment of fibrinopeptide A, Ala-Asp-Ser-Gly, demonstrated very effective coordination of N-terminal ‘residues to Cu(I1) as a result of the @carboxylate group of the Asp residue in the second position [l]. This fragment could model only the metal binding power of the N-terminal residues and so assumes the absence of steric constraints from the spatial organization of the hexadecapeptide and of competitive coordination to amino acid residues further along the chain. These assumptions appear reasonable but require confirmation by a study of the naturally occurring ligand. We therefore report the synthesis of fibrinopeptide A and the
Address correspondence Leeds LS2 9JT, U.K. Journal of Inorganic 0
to: Dr. Leslie D. Pettit, Department
Biochemistry,
1991 Elsevier Science Publishing
of Inorganic
Chemistry,
The University,
149
44, 149- 153 (1991)
Co., Inc., 655 Avenue of the Americas,
NY. NY 10010
0162-0134/91/$3.50
150
J-F. Galey et al.
results Cu(II)
of potentiometric ZniII).
and spectroscopic
studies
of the complexc~
formed
with
and
EXPERIMENTAL Peptide
Synthesis Fihrinopeptide A was synthesised using Merrifield solid phase synthetic methods based on 1% crosslinked chloromethylated resin (Novabiochem! 121. The staring material, resin-Boc-Arg-Tos (Tos -=-roluene-4-suphonatc). ~a’\ preparcd from the resin and Boc-At-g-Tos b> the cebium salt method [?I; amino ac& ilfloq~rotectcd~ were obtained commcrciallq iNovabiochem. Propeptide (II’ Bachcn,i Rcacti\ t‘ \itlt: chains were protected as follows: Arg by the To> group: llsp’ :irld (;i\?’ ’ 11~the cyclohexyl group because these residues follow Ala and protection uGrt3 cyclohe\~l minimizes /S--migration: Asp-‘, GILI'. and Ser by the henqi group. i‘icavagc ~ti peptide from the resin and cieavage of protecting b‘croup\ from Ihi: pilptide i\r"rC performed using HF in the prcsencc of paracrrso!. T’hc r1:huiting hcu&,apepridc was purified by gel filtration i-I?X gel) and purity was chci,i;c*il b>’ ~hr-l~Il~aiogl-;tl~~~ (paper and HPLC) and b> potentiometry. Amino ai.ici ana1y \ I* liar ,r~crf~rrrm.xl‘IiTCI peptide hydrolysis with h mol dm ’ HC’1 using a Beckman ‘W(i ;!r~~l\~/~r and i:atc the results: Asp, 1.Ol: Ser. 0.81: Glu. 2.06: Gly. 5.01. ,Aia. J.01 1 V) cpcctra 0:3 .I Mvlarh III Jobin-Yvon dichrograph ill the 220--800 nm region. .411 CD rc~llt~ arc c\-pn’+cd in terms of 14~ =:-f1 -- tr. Electron paramagnetic resonance CEPR) spectra were recorded ton a I‘arian El02 spectrometer at liquid nitrogen temperature at 9. 13 ciIJ/, Potentiometric
Studies
Stability constants for H ’ . Cu’ ’ . and Zn’ ’ complexes were calculated from titration curves carried out at 25 “C using total volumes of 1.S cm ‘. Alkali was added from a 0. I cm’ micrometer syringe which had been calibrated by both weight [Itration ;md the titration of standardized materials. Experimental details were: peptide concentration. 0.0025 mnl dm ‘; copper concentration, 0.001 -0.002 tnol dm ‘: ionic strength. 0. 10 mvl dm ’ (KNO,): pH range for complexation,
