Solvent Effects on Luteinizing‐ and Follicle‐Stimulating‐Hormone Releasing Factor Polymorphism Studied by Circular Dichroism

Abstract

Luteinizing-and follicle-stimulating-hormone releasing factor has been studied by circular dichroism and compared with two of its peptidic fragments. Aqueous solutions of the decapeptide have been examined as a function of pH and temperature. The hormone seems to exist under a random conformation in acid medium. Increasing the pH or heating in neutral medium, favor a shift towards ordered structures. Dissolving the hormone solutions with different ratios of trifluoroethanol/water or in hexafluoroisopropanol lead to a more dramatic change in the spectra : ordered conformations are predominant for high trifluoroethanol concentrations. From our observations, it is suggested that two types of peptidic chromophore interactions are responsible for the hormone structures in the alcoholic solvents mentioned above. The role of the environment upon the conformation(s) adopted by the decapeptide is also discussed.The luteinizing hormone and follicle-stimulatinghormone releasing factor is a decapeptide isolated from bovine and porcine hypothalami [1,2]. Its amino-acid sequence is known as : <Glu-His-Trp-SerTyr-Gly-Leu-Arg-Pro-Gly(NH,) [3] and the peptide has been synthesized [4--81 as well as a large number of analogs [9-111. The biological activities of the analogs have been compared to those of the natural decapeptide. These data have led to speculations with respect to the possible conformations of the releasing factor [lo-121. To understand the relationship between primary structure and biological activities one has to gain insight into the parameters needed both for the specific binding and for the triggering of the biological response. The preparation of tritium-labelled releasing factor was a first step towards this goal [13,14] and permitted the existence of two pituitary binding sites [13] to be shown.A further step corresponds to conformational studies of the releasing factor in solution. As the latter is a linear decapeptide its conformational degrees of liberty are potentially quite large. It is, however interesting to study the restrictions imposed on the conformations of the releasing factor by different surrounding conditions. Correlations between changes in the environment and changes in the peptide primary structure on its conformational features might thus be established and eventually set in parallel with biological responses.With this perspective in mind the luteinizing hormone and follicle-stimulating-hormone releasing factor has been studied by circular dichroism. Aqueous solutions a t different pH and a t various temperatures as well as organic solutions of the peptide and two peptidic fragments were examined. Trifluoroethanol and hexafluoroisopropanol promoted a marked stabilization of ordered conformation( s). MATERIALS AND METHODSLuteinizing-and follicle-stimulating-hormone releasing factor, its N-terminal tripeptide and the C-terminal heptapeptide, synthesized via the classical approach of peptide synthesis [S] were generously supplied by Drs R. 0. Studer and D. Gillesen, (Hoffman-la Roche, Basel...

Circular dichroism study of the solution conformation of luteinizing hormone releasing hormone

“…In water at 27 °C the spectrum shows a positive band at 229 nm and a well-defined, negative shoulder at 217 nm. Raising the temperature to 65.5 °C causes the appearance of a negative band at 237 nm and, in agreement with Marche et al (1973), loss of the positive band and intensification of the 217-nm shoulder. Changing the solvent from water to a 90% dioxane-water mixture causes the appearance of a negative band at 236 nm with loss of the positive band and the appearance of a negative band at 219 nm.…”

“…(a) intensification of the CD contribution of the C-terminal pentapeptide moiety (which overall is negative at elevated temperatures) without loss of a local maximum nor change in its position and (b) diminution of the amplitude of the His band. In other words, our results indicate that the growth of the shoulder with temperature is not to be interpreted in terms of a trend toward ordered structures as proposed by Marche et al (1973) but rather to depopulation of conformers which show a positive band at 218 nm and to a change in the CD of His. Changing the solvent to a 90% dioxane-water mixture causes the appearance of a distinct negative band at 219 nm.…”

“…Our interest in the CD of LHRH stems from the observation that, whereas in aqueous solution its spectrum over the wavelength range 260-225 nm is dominated by positive aromatic bands (Marche et al, 1973;Mabrey & Klotz, 1976; curve a in our Figure 1A), the spectrum of the C-terminal pentapeptide fragment of LHRH, Gly6-Leu7-Arg8-Pro9-Gly10-NH2, exhibits a relatively weak negative band with an extremum at 231 nm (curve b in Figure 1 A). The questions posed are "Is this negative band a spectral component of LHRH itself but masked by overlying aromatic contributions?"…”

“…Before proceeding to these measurements, however, it is necessary in the way of background to reiterate the findings of other investigators with regard to the effects of the structure-promoting solvent trifiuoroethanol (Marche et al, 1973;Mabrey & Klotz, 1976) and the structure-disrupting solvent trifluoroacetic acid (Mabrey & Klotz, 1976) on the far-ultraviolet CD of LHRH. Whereas highly ordered conformers (which have been ascribed to helical and/or ß structures) predominate in trifiuoroethanol (curve c of Figure 1 A), they are virtually disrupted in a 20% trifluoroacetic acid-80% trifiuoroethanol mixture (curve d in Figure 1A).…”

Conformation of gonadotropin releasing hormone

“…Furthermore, the similarity in behavior of the active peptide and its inactive analogues indicates that biological effectiveness is not associated with a preferred conformation of the hormone in solution. Similar conclusions have been reached recently by Marche et al (1973). Comparable behavior has also been observed for the nonapeptide hormone bradykinin for which circular dichroism studies reveal a flexible molecule possessing several different conformations (Cann et al, 1973).…”