ISBN: 978-83-60043-10-3 - eurobic9

Eurobic9, 2-6 September, 2008, Wrocław, Poland
P28. Complexes of Histidine Analogues of Oxytocin and Vasopressin with
Zn(II) and Cu(II) ions Studied by ESI-MS Mass Spectrometry
M. Cebrat, A. Sochacka, P. Stefanowicz
Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383 Wroclaw, Poland
e-mail: lukasz@wchuwr.pl
Oxytocin (OT) and arginine-vasopressin (AVP) belong to a group of neurohypophysial peptide hormones
present in virtually all vertebrates and many other species. They are nonapeptides with a disulfide bridge
between Cys residues 1 and 6. OT is associated with reproductive functions, stimulation of uterine contractions
during labor and milk ejection during lactation, whereas AVP facilitates water reabsorption by the kidney and
the contraction of smooth muscle cells in arteries. Divalent metal ions appear to be important element of the
OT/AVP system [1].
Some extracellular proteins employ disulfide bridges to stabilize topologies that are similar to the intracellular
zinc-stabilized motifs [2, 3]. Therefore, we decided to check whether it is possible to retain the conformation and
the biological activity of OT and AVP peptides by replacing the disulfide bond in the Cys-Cys pair by the His-
M-His complex (M = metal ion). The binding abilities of the His-analogue of AVP towards Cu(II) were also
studied by potentiometric techniques [4]. Here we report the formation and fragmentation pattern of the
complexes of the OT and AVP analogues with both Cys residues substituted by His with Zn(II) and Cu(II) ions
as observed by ESI high resolution mass spectrometry:
1. Ac-His-Tyr-Phe-Gln-Asn-His-Pro-Arg-Gly-NH2 [His 1,6 ]AcAVP OS-1
2. H-His-Phe-Phe-Gln-Asn-His-Pro-Arg-Gly-NH2 [His 1,6 ,Phe 2 ]AVP OS-2
3. Ac-His-Phe-Phe-Gln-Asn-His-Pro-Arg-Gly-NH2 [His 1,6 ,Phe 2 ]Ac-AVP OS-3
4. H-His-Tyr-Ile-Gln-Asn-His-Pro-Leu-Gly-NH2 [His 1,6 ]OT OS-4
5. Ac-His-Tyr-Ile-Gln-Asn-His-Pro-Leu-Gly-NH2 [His 1,6 ]Ac-OT OS-5
6. His-Phe-Ile-Gln-Asn-His-Pro-Leu-Gly-NH2 [His 1,6 ,Phe 2 ]OT OS-6
7. Ac-His-Phe-Ile-Gln-Asn-His-Pro-Leu-Gly-NH2 [His 1,6 ,Phe 2 ]Ac-OT OS-7
8. H-His-Tyr-Phe-Gln-Asn-His-Pro-Arg-Gly-NH2 [His 1,6 ]AVP OS-8
At pH 6-7 (NH4Ac-buffered peptide solution) a complexes of a type [peptide +M] 2+ are visible in all recorded
mass spectra. The intensity of the peak is rather low as compared to [peptide +2H] 2+ and significantly lower in
case of Zn(II) than for Cu(II) complexes. During MS/MS fragmentation of this complex a respective C-terminal
tripeptide Pro-Aaa-Gly-NH2 (Aaa = Arg or Leu) is easily cleaved while the metal ion is retained by the Nterminal
fragment. Further fragmentation results in subsequent cleaving of the amino acid residues mostly from
the C-terminus of the peptide. Typicaly yn ions are formed during this process, but xn fragments are often
observed as well.
Of the two His residues, His 1 seems to chelate metals more efficiently. Even in the case of the acetylated
peptides we can see N-terminal tripeptide fragments containing Cu (Zn) ions.
Acknowledgement: ESI MS and MS/MS spectra were recorded on Bruker apex ultra 7T FTMS mass
spectrometer in the Mass Spectrometry Laboratory at the Faculty of Chemistry, University of Wroclaw.
References:
[1] T. Wyttenbach, D. Liu, and M.T. Bowers, J. Am. Chem. Soc., 130, 5993 (2008).
[2] C.A. Orengo, J.M. Thornton, Structure, 1, 105 (1993).
[3] I.Z. Siemion, Z. Szewczuk, Wiadomości Chemiczne, 45, 755 (1995).
[4] J. Brasuń, M. Cebrat, A. Sochacka, O. Gładysz, J. Świątek-Kozłowska, accepted by Dalton Trans.
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