Proton Magnetic Resonance Comparison of Neurohypophyseal Hormones and Analogs: Deletion of Amino Groups and the Conformation of Lysine Vasopressin

Abstract

Proton nuclear magnetic resonances of two analogs of the mammalian antidiuretic hormone, lysine vasopressin, have been assigned. The analogs studied were deamino-lysine vasopressin and deamino-8-tosyllysine vasopressin, formally derived from lysine vasopressin by removal of the potential cationic sites. Most assignments made in deuterated dimethylsulfoxide at 220 MHz were directly derived from the already determined spectrum of lysine vasopressin in this solvent [Walter et al., Proc. Nat. Acad. Sci. USA (1972) 69, 1920]. Comparison of chemical shifts of peptide NH peaks, aCH-NH coupling constants, temperature dependence of peptide NH resonances, and proton-deuterium exchange rates revealed that from a conformational standpoint both deamino analogs occupy intermediary positions between lysine vasopressin and oxytocin.From the point of view of conformation, the most significant difference between the congeneric neurohypophyseal hormones, oxytocin and lysine vasopressin (LysVP), is the greater hydrophilicity of LysVP. In addition to the NH2-terminal amino group of Cys-1 that is present in both of these nonapeptide hormones, LysVP possesses a second potential cationic site in the e-amino group of the lysine residue in position 8. In order to evaluate the extent to which the presence of two amino groups in LysVP contributes to its conformational variances with respect to oxytocin (1-3), we have compared the proton magnetic properties of these two hormones with those of the two LysVP analogs, [1-f-mercaptopropionic acid, 8-lysine]-vasopressin (also referred to as deamino- Fig. 1), in which the number of primary amino groups has been reduced stepwise. In d-NH2LysVP the NHrterminal amino group is formally replaced by a hydrogen atom, and in dNH2TosLysVP an additional modification is introducedthe Camino group of Lys is tosylated. MATERIALS AND METHODSd-NH2LysVP and d-NH2TosLysVP were prepared by Havran (manuscript in preparation),-who used a solid-phase method of peptide synthesis (4, 5). This d-NH2LysVP exhibited a rat pressor activity (6) of 132 ± 7 U/mg, which is identical to the activity of 126 d-2 U/mg reported for this analog when it was prepared by conventional methods of peptide synthesis (7).Spectra were recorded on a Varian Associates HR-220 NMR Spectrometer, and proton double-resonance experiments were done as described (3) (3) Abbreviations: Me2SO, dimethylsulfoxide; Me4Si, tetramethylsilane; LysVP, lysine vasopressin;

Conformational Studies of Oxytocin, Lysine Vasopressin, Arginine Vasopressin, and Arginine Vasotocin by Carbon-13 Nuclear Magnetic Resonance Spectroscopy

“…Investigation of neurohypophyseal hormones by '3C NMR spectroscopy promises, therefore, to yield valuable information in extending past conformational studies of these peptides by thin-film dialysis (39), chromatographic methods (40), circular dichroism (41), minimization energy calculation (42), and proton NMR (1)(2)(3)(4)(5)(6)(7)(43)(44)(45)(46)(47) (31,48,49) and D20 (31,48) (4, 7). In making the assignments it was assumed that the order of 'IC resonances of the hormones was the same as in the acyclic nonapeptide for all residues except cysteine.…”

“…The spectra of all four hormones, which can be viewed as structural analogs of each other, were then inspected for internal consistency of assignments. This approach was efficient and reliable for the assignment of neurohypophyseal hormones by proton resonance spectroscopy (7,45) Having completed the assignments of all carbons of the hormones and discussed spectral aspects and conformational implications, it was of interest to investigate the effect of progressive changes of hydrogen-ion concentration. As the pH was increased stepwise from 2.6 to 8.3, two major effects were observed with all hormones.…”

Proton Magnetic Resonance Study of Peptide Conformation: Effect of Trifluoroethanol on Oxytocin and 8-Lysine-Vasopressin

“…This procedure was desirable since PMR assignments of oxytocin (13), [Lys8]-vasopressin (16,19,23), and its analogs (20) had been made in Me2SO and since neurohypophyseal hormones polymerize in MeOH (unpublished). Changes in chemical shifts of the PMR resonances of the amide NH and aromatic CH protons of these hormones were followed as Me2SO was progressively enriched with FXE (Fig.…”

“…Thus the rate of hydrogen replacement, a criterion sensitive to kinetic parameters, reflects in the exchange process the rate-limiting step, which may be either the unfolding of the peptide or the actual displacement of the exposed peptide hydrogen (2). Moreover change properties, temperature dependencies of their amide proton resonances, and other conformational features we have previously described (13)(14)(15)(16)(17)(18)(19)(20)(21). Independent PMR work on [Lys8]vasopressin is also being done in other laboratories (22)(23)(24)(25).…”

“…Resonances (19,20,26). Specifications of the Varian HR-220 NMR spectrometer have also been described (19).…”