Milan Kodíček

Heat denaturation of human orosomucoid in water/methanol mixtures

Heat denaturation of orosomucoid in solutions of methanol concentrations ranging from 0 to 70% (v/v) has been studied by using circular dichroism, intrinsic protein fluorescence and thermal difference absorption spectroscopy. Regardless of its high saccharide content (40%), the highly cooperative denaturation transition of orosomucoid is fully reversible in neutral water solution. A two-state model has been successfully applied; the numerical analysis results in thermodynamical parameter values that are in close agreement with previously reported experimental data from calorimetric measurements. However, in solutions containing even minute concentrations of methanol (5%) the heat denaturation is irreversible. After cooling of the denatured protein the refolded molecules exhibit a higher alpha-helical content than the native one. Possibilities of methanol interaction with native and denatured protein molecule are discussed.

On the molecular conformation of human haemopexin: I. Reactivity of the tyrosine and tryptophan side chains

The reactivity of the aromatic side chains of Tyr and Trp in human haemopexin were studied by chemical modifications and analysis of spectrophotometric titration curves. It has turned out that: 1. Under non-denaturing conditions the aromatic rings of Tyr resisted both acetylation and nitration. 2. Three indole groups of Trp reacted with the Koshland agent, without the native conformation of the protein being markedly affected (CD spectra). 3. Oxidation by N-bromosuccinimide split the peptide chain and the molecular conformation collapsed. 4. The Tyr residues could be placed into three classes, according to their pK values: 2 (or 1 in the haem-haemopexin complex) were normally accessible to titration, 5 were masked and the remaining 7 (or 8) were buried. 5. The spectrophotometric titration curve could not be analysed in terms of the Linderstrøm-Lang equation. The findings 1 to 3 refer to both haemopexin and its complex with haem; the spectrophotometric titration curves of the two molecules are very similar too. Consequently, the binding of haem is not associated with a profound alteration of the molecular architecture. The generally low reactivity of the side chains studied indicates that the hydrophobic peptide core of this glycoprotein is a compact one, very restricted in its contacts with the environment.

On the molecular conformation of human haemopexin. II. Analysis of circular dichroic spectra.

The circular dichroic (CD) spectra of haemopexin in the far ultraviolet region exhibit an atypical positive maximum at 231 nm, which prevents determination of the secondary structure by the usual methods. We have developed a modified analytical method by which it was possible to calculate the secondary structure of the protein (8% of alpha-helix, 10% of beta-conformation) and the actual wavelength (229 nm) and intensity of the above-mentioned positive band. Measurements and analysis of the CD spectra of haemopexin in an alkaline medium gave information ascribing a major part of intensity of the band at 229 nm to the B1u transition of Tyr; we believe that some other electronic transitions contribute to this band, too. An assignation of the individual extremes of the CD spectrum in the near ultraviolet region to the individual electronic transitions is propounded. The dichroic bands of Trp in this region and the temperature dependence of the CD spectra in the far ultraviolet region corroborate our idea that the haemopexin molecule contains a very compact hydrophobic peptide core. Similarities of the CD spectra of haemopexin to those of the haem. haemopexin complex throughout the ultraviolet region suggest a conformational likeness of these two molecules.

Perfluoroalkylated derivatives of 6-deoxy-6-ethylamino-d-galactose, 1-deoxy-1-methylamino-d-glucitol, and 1-amino-1-deoxy-d-glucitol: syntheses, hemocompatibility, and effect on perfluorocarbon emulsion

N-polyfluoroalkyl derivatives of 6-deoxy-6-ethylamino-1,2;3,4-di-O-isopropylidene-alpha-D-galactopyranose (8-10), 1-deoxy-1-methylamino-D-glucitol (13-15), and 1-amino-1-deoxy-D-glucitol (16-18), all possessing perfluoroalkyl segment, were prepared using nucleophilic epoxide ring opening of 2-[(perfluoroalkyl)methyl]oxiranes 1-3. Co-emulsifying properties and hemolytic activity of the new perfluoroalkylated amphiphiles were tested. Both types of the polyol derivatives 8-10 and 13-18 generally displayed good to excellent co-emulsifying properties on testing on perfluorodecalin/Pluronic F-68 microemulsions. Mono-perfluoroalkylated compounds 8-10 and 13-15 displayed high hemolysis, whereas acyclic bis-perfluoroalkylated compounds 16-18 were non-hemolytic even for short perfluorobutyl segment (16). The properties were generally improving with increasing perfluoroalkyl chain length.

The conformation and stability of human Zn-α2-glycoprotein in aqueous and methanolic solutions

Abstract The stability of Zn-α2-glycoprotein has been studied using UV-spectroscopy and circular dichroism with respect to the influence of temperature, pH, and solvent composition. It has been found that: (1) this protein contains a relatively high proportion of β-sheet (60%) and a very low amount of other periodic structures as estimated from circular dichroic spectra: (2) at pH 7.4, the circular dichroic spectra change reversibly in the temperature range between 25 and 85°C; small disturbances were observed at 265 nm; (3) with the assumption of the two-state process, the temperature of cooperative denaturation was Tm = 66°C, vant Hoffs enthalpy of this process was 27 kJ mol−1; (4) up to pH 9.5 the dichroic spectrum appeared the same as at pH 7.4; (5) in the presence of methanol (vol. fraction 50%), no isodichroic points on the circular dichroic spectra were found during temperature denaturation; after cooling from 85°C, the α-helix content was higher than in the native protein; (6) in the molecule of Zn-α2-glycoprotein, 14 out of 18 tyrosines can dissociate with the instrinsic pK = 11.2; and (7) the temperature perturbation difference spectra yielded nonlinear ΔA vs. T curves with temperature transition corresponding to the values found in the circular dichroic spectra; the numbers of chromophores exposed to the solvent as determined by the temperature difference spectra were: 4 tyrosines, 1 tryptophan, and 1 phenylalanine. In several aspects, a parallel has been found between Zn-α2-glycoprotein and orosomucoid (acid α1-glycoprotein), another plasma glycoprotein.