Separation and investigation of structure–mobility relationship of gonadotropin-releasing hormones by capillary zone electrophoresis in conventional and isoelectric acidic background electrolytes

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Abstract

Capillary zone electrophoresis (CZE) has been applied to qualitative and quantitative analysis, separation and physicochemical characterization of synthetic gonadotropin-releasing hormones (GnRHs) and their analogs and fragments. Structurally related peptides were separated in conventional and isoelectric acidic background electrolytes (BGEs), pH 2.18–2.50. Best separation was achieved in isoelectric BGE composed of 200 mM iminodiacetic acid, pH 2.32. The effective electrophoretic mobilities, mep, of GnRHs in five BGEs were determined and four semiempirical models correlating effective mobility with charge, q, and relative molecular mass, Mr, (mep versus q/Mrk, where k is related to the molecular shape) were tested to describe the migration behavior of GnRHs in CZE. None of the models was found to be quite definitively applicable for the whole set of 10 GnRHs differing in size (tetrapeptide–decapeptide) and positive charge (0.91–3.00 elementary charges). Nevertheless, for the dependence of mep on q/Mrk, the highest coefficient of correlation, R = 0.995–0.999, was obtained for k close to the value 0.5 in all five acidic BGEs. This indicates that the most probable structure of GnRHs in these BGEs can be predicted as a random coil.

Introduction

Gonadotropin-releasing hormone (GnRH) is a peptide neurohormone responsible for the release of follicle stimulating hormone and luteinizing hormone from the anterior pituitary (adenohypophysis). It is present in different biological species such as human, pig, lamb, chicken, sea bream and salmon with small modifications in its peptide sequence. Human GnRH, hGnRH, alternatively termed luteinizing hormone-releasing hormone (LHRH), is a decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), which is synthesized in neurosecretory cells within the hypothalamus and regulates reproductive functions and maintenance of secondary sex characteristics in males and females. LHRH agonists are used for the treatment of hormone-dependent breast and prostate cancers [1]. In reproductive medicine, they serve to prevent a premature LH surge prior to stimulation of ovulation. Synthetic hGnRH is produced under the name gonadorelin. Several commercially available analogs are used as drugs. Buserelin, [Des-Gly10,d-Ser(tBu)6,Pro-NHEt9]hGnRH, and triptorelin, [d-Trp6]hGnRH, are used in treatment of prostate cancer, deslorelin, [Des-Gly10,d-Trp6,Pro-NHEt9]hGnRH, for treatment of true precocious puberty, goserelin, [d-Ser(tBu)6,Azagly10]hGnRH for treatment of advanced breast cancer and nafarelin, [d-2-Nal6]hGnRH, in treatment of endometriosis.

Synthetic GnRHs and their analogs and fragments are frequently used as model analytes, usually in mixtures with other peptides, to demonstrate new methodology and/or instrumentation developments and optimization of separation conditions for analysis and separation of peptides by capillary electromigration methods [2]. Successful combination of capillary electrophoresis (CE) with mass spectrometry (MS) detection without make-up flow or nebulizing gas was shown by analysis of gonadorelin with sensitivity on the level of immunoassay [3]. Home-made on-line preconcentration CE based on the coupling of capillary packed with C18 sorbent to the fused silica capillary was optimized for the purity control of synthetic biologically active peptides including GnRH in BGE composed of 25 mM K2HPO4, pH 3.5 [4]. Membrane preconcentration CE-MS/MS was appropriately constructed for sequencing biologically active peptides at the sub-100 fmol level [5]. The analysis of nine model peptides was performed in 2 mM ammonium acetate and 1% (v/v) acetic acid. Successful application of fused silica (FS) capillary modified by positively charged alkylaminosilyl monomer to capillary zone electrophoresis (CZE) and capillary electrochromatographic (CEC) separations of the mixture of peptides and proteins was presented using UV-absorption and MS detection [6]. Rapid separation of five peptides including GnRH was achieved in 5 mM acetic acid in 50% (v/v) MeCN. A new CE-nanoflow electrospray ionization (ESI) interface, where separation column, an electrical porous junction and spray tip were integrated on single FS capillary, was found as a suitable device for analysis and separation of nine peptides including GnRH and proteins in 1 M acetic acid, pH 2.4 [7]. Human GnRH and its four analogs, [d-Ala6]hGnRH, [d-Lys6]hGnRH, [d-Phe2,d-Ala6]hGnRH and [Gly-OH10]hGnRH were analyzed in capillary derivatized with 3-(aminopropyl)trimethoxysilane in 0.01 M acetic acid, pH 3.5, by CE with ESI-MS detection [8]. For determination of side products of buserelin synthesis, CE with field-amplified sample injection was used [9] and for determination of purity degree and counter-ion content in lecirelin, [d-Tle6,Pro-NHEt9]GnRH, CZE and capillary isotachophoresis were applied [10]. Optimal separation conditions were found in acidic BGEs with pH less than 3.5. An off-line coupling of CE and MALDI-MS was successfully applied for the analysis of four standard peptides, proteins and real tear fluid in 50 mM ammonium acetate buffer, pH 7.4 [11]. According to the known or established characteristics, such as dissociation constants of ionogenic groups of therapeutic peptides optimal separation conditions were determined for set of seven peptides including buserelin and triptorelin for CE [12]. The conditions were tested in a wide pH range 2–12; the best separation was achieved at pH 2.85, in BGE composed of 50 mM acetic acid and 50 mM formic acid, pH adjusted by NH4OH. The same set of peptides was successfully applied for separation and characterization in hydroorganic mixture of formic acid, acetic acid and 2-propanol by CE-ESI-MS with commercial [13] and home-made graphite coated sheath-flow interface [14] and as well for comparison of predicted and experimentally obtained resolution, electrophoretic mobility and retention factor in CE and HPLC [15]. Complete separation of gonadorelin and its five analogs was provided by RP-HPLC in mobile phase consisting of MeCN and phosphate buffer, pH 2.5, by CE in phosphate or borate buffer and as well by micellar electrokinetic chromatography (MEKC) with the same BGE with addition of cetyltrimethylammonium bromide (CTAB), 3-[(3-choloamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and Triton X-100 as micellar constituents [16]. Alternative detection technique to UV-absorption, contactless conductivity detection was employed for the analysis of mixture of nine peptides including GnRH in phosphate buffer, pH 2.5 [17]. Detection limits at the μM level in combination with good resolution were shown. Native GnRHs are generally used to study biological regulatory processes such as kinetics and mechanism of their action. A rapid CE assay for measuring the stability of human and salmon GnRH in the presence of intestinal enzymes was developed and validated [18]. The analysis was performed in acetic acid-based BGE, pH 4.0, and applied to the stability of GnRH analogs in salmon intestinal digest. Determination of gonadorelin in plasma by on-capillary preconcentration CE system was demonstrated [19]. For suppressing adsorption on the wall, the capillary had cationic coating and for increasing sensitivity and selectivity MS detection was used.

The aim of this work was to perform qualitative and quantitative analysis of synthetic preparations of human, salmon and chicken GnRHs, and their analogs and fragments, by CZE in acidic conventional and isoelectric BGEs. Suitable experimental conditions should be developed for CZE separation of the mixtures of these structurally related peptides. In addition to the purity degree also some physicochemical characteristics of the analyzed peptides, such as effective electrophoretic mobilities of GnRHs at standard temperature, 25 °C, should be determined, and different models of the dependence of mobility of GnRHs on their charge and size should be tested in order to predict the probable structure of GnRHs in solution.

Section snippets

Models of correlations between mobility of peptides and their charge and size

Several semiempirical models correlating effective electrophoretic mobilities of peptides, mep, with their effective charge, q, and molecular size expressed as relative molecular mass, Mr, or number of amino acids in polypeptide chain, n, respectively, have been developed. The models are based on Stokes law, describing the motion of a particle in liquid medium, and on the action of electric field force on charged molecule.

Offord [20] has firstly quantitatively described this relationship for

Chemicals

All chemicals used were of analytical reagent grade. Iminodiacetic acid (IDAA) was obtained from Bachem (Bubendorf, Switzerland), phosphoric and acetic acids were obtained from Lachema (Brno, Czech Republic) and Tris (tris(hydroxymethyl)aminomethane) was supplied by Serva (Heidelberg, Germany). Isophorone (3,5,5-trimethyl-2-cyclohexen-1-one) was supplied by Fluka (Buchs, Switzerland).

Peptides

The list of analyzed peptides and their abbreviations, sequences and relative molecular masses, Mr, are

Selection of separation conditions and determination of peptide charge

The strategy for the rational selection of experimental conditions for CZE analysis and separations of GnRHs and their analogs and fragments followed the general rules of selection of suitable CZE separation conditions [34] and took into account the specific properties of these peptides resulting from their structure. The selection of the composition of the BGEs includes the type and concentration of buffer components and pH, and it also takes into account the requests for solubility and

Acknowledgements

This work was supported by the Grant Agency of the Czech Republic, grants no. 203/04/0098, 203/05/2539, 203/06/1044, and by the Czech Academy of Sciences, research project Z40550506. We thank to Mrs. V. Lišková for her skilful technical assistance and Dr. D. Koval for his help in preparation of this manuscript.

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