Spectroscopic properties of Carcinus aestuarii hemocyanin and its structural subunits

Hemocyanin (Hc) of Carcinus aestuarii contains three major and one minor electrophoretically separable polypeptide chains which were purified by fast protein liquid chromatography (FPLC) ion exchange chromatography. N-terminal amino acid sequences of four structural subunits (SSs) from C. aestuarii were compared with known N-terminal sequences from other arthropodan hemocyanins. The conformational changes, induced by various treatments, were monitored by far UV, CD and fluorescence spectroscopy. The critical temperatures for the structural subunits, Tc, determined by fluorescence spectroscopy, are in the region of 52-59 degrees C and coincide with the melting temperatures, Tm (49-55 degrees C), determined by CD spectroscopy. The free energy of stabilization in water, delta GDH2O, toward guanidinium hydrochloride is about 1.3 times higher for the dodecameric Hc as compared to the isolated subunits and about one time higher for Cal, comparing with other SSs. The studies reveal that the conformational stability of the native dodecamer towards various denaturants (temperature and guanidinium hydrochloride) indicate that the quaternary structure is stabilized by oligomerization between structural subunits, and the possibility of a structural role of the sugar mojeties cannot be excluded.     

Hydrophobic interaction chromatography coupled with atomic fluorescence spectrometric detection Effect of the denaturation on the determination of thiolic proteins

Hydrophobic interaction chromatography coupled online with chemical vapour atomic fluorescence spectrometry (HIC-CVGAFS) has been optimized for the analysis of thiolic proteins in denaturing conditions. Proteins are pre-column simultaneously denatured and derivatized in phosphate buffer solution containing 8.0 mol dm⁻³ urea and p-hydroxymercurybenzoate (PHMB) and the derivatized denatured proteins are separated on a silica HIC Eichrom Propyl column in the presence of 8.0 M urea in the mobile phase. Post-column online reaction of derivatized denatured proteins with bromine, generated in situ by KBr/KBrO₃ in HCl medium, allowed the fast conversion of the uncomplexed PHMB and of the PHMB bound to proteins to inorganic mercury also in presence of urea. Hg²⁺, present in solution as Hg²⁺-urea complex, is selectively detected by AFS in a Ar/H₂ miniaturized flame after sodium borohydride reduction to Hg. Under optimized conditions, online bromine treatment gives a 100±2% recovery of both free and protein-complexed PHMB. Denatured glyceraldehyde-3-phosphate dehydrogenase, aldolase, lactate dehydrogenase, trioso phosphate isomerase and β-lactoglobulin have been examined. As the sensitivity and limit of detection of proteins in the HIC-CVGAFS apparatus depends on number of SH groups reacting with PHMB, the denaturation process, which increases the number of PHMB-reactive thiolic groups in proteins, improves the analytical performances of the described system in protein analysis. The detection limit for the denatured proteins examined was found in the range of 10⁻¹⁰-10⁻¹² mol dm⁻³, depending on the considered protein, with linear calibration curves spanning over four decades of concentration.     

Gelation of β-lactoglobulin in the presence of propylene glycol alginate: kinetics and gel properties

The role of the non-gelling polysaccharide, propyleneglycol alginate (PGA), on the dynamics of gelation and gel properties of β-lactoglobulin (β-lg) under conditions where the protein alone does not gel (6%) was analyzed. To this end, the kinetics of gelation, aggregation and denaturation of β-lg in the mixed systems (pH 7) were studied at different temperatures (64–88 °C). The presence of PGA increased thermal stability of β-lg. The rate of β-lg denaturation was decreased and the onset and peak denaturation temperatures increased by 2.2–2.4 °C. PGA promoted the formation of larger aggregates that continued to grow in time. An average aggregate diameter of approximately 300 nm is reached at the gel point in the mixed β-lg+PGA systems, irrespective of the heating temperature. Comparing the activation energies for the aggregation (193 kJ/mol), denaturation (422 kJ/mol) and formation of the primary gel structure (1/t_gel) (256 kJ/mol) processes in the mixed protein–polysaccharide system, it can be concluded that the rate determining step in the formation of the primary gel structure would be the aggregation of protein. E_a values for the processes after the gel point (solid phase gelation) suggest a diffusion limited process because of the high viscosity of the solid gelling matrix. The characteristics of the mixed β-lg+PGA gels in terms of rheological and textural parameters, water loss and microstructure were studied as a function of heating temperature and time. The extent of aggregation and the type of interactions involved, prior to denaturation seem to be very important in determining the gel structure and its properties.     

德国小蠊变应原Bla g 2蛋白变复性的荧光光谱研究

包涵体中的重组蛋白经抽提后可以在变性状态下纯化,而纯化后的复性过程是基因工程下游处理的重要环节。通过对变应原Bla g 2蛋白复性前后荧光光谱的比较和分析、变性剂(尿素和SDS)对复性后Bla g 2蛋白的荧光滴定实验、以及复性后Bla g 2在不同pH下的荧光光谱分析,推断出Bla g 2蛋白分子在不同环境下构象的变化及其光谱学特征,初步建立了一种新的检测重组变应原蛋白变复性的光谱实验方法。     

Effect of water content on electrical conductivity of fish skin collagen

The increased interest in fish skin collagen results from the risk of BSE disease and the production of native collagen by the acidic hydration method. As a material, FSC derived by the novel acidic hydration method was used. The bovine Achilles tendon (BAT) collagen type I was applied as a control material. Measurements were carried out at the temperature range 2980 K-510 K. Each sample was heated twice. During the first heating run for FSC, to 380 K, the maximum was localized at 343 K, at electrical conductivity of 7 · 10⁻⁶ S/m. In the second heating run to 510 K, the maximum was observed at 443 K for FSC, and at 487 K for BAT collagen. The peaks revealed in the temperature range 320-350 K were related to the free water and bound water release, alike for FSC and BAT collagen. The process of water removal for both types of collagen takes place in similar temperature ranges.     

Torsion and entropy driven denaturation of DNA

A unified theory of the denaturation transition having torsion energy as the control parameter has been formulated here in the framework of the mapping of a DNA molecule onto a Heisenberg spin system. The torsion energy incorporates the torque, tension and temperature, the latter being associated with the twist angle. The denaturation transition can be mapped onto the quantum phase transition induced by a quench when the temperature effect is incorporated in the quench time and torsion takes the role of the external field. The denaturation transition occurs when the entanglement entropy of the spin system vanishes.     

Assessment of collagen-based materials which are supports of cultural and historical objects

The thermal degradation of collagen-based materials, viz. collagen, recently manufactured parchments and tanned leathers, a heritage parchment and patrimonial (historical) leathers, was investigated by thermal analysis methods. With progressive heating, all these materials exhibit three main successive processes, associated with dehydration, protein denaturation and pyrolysis/oxidation. The analysis of both denaturation and thermo-oxidation revealed significant differences between aged leather, collagen, recently manufactured parchments and tanned leathers in terms of the number of overlapping processes (reflected in the number of peaks recorded at denaturation) and of the oxidative degradation rates, given by the normalised curves of differential thermal gravimetry (DTG). The various observations are proposed as qualitative indices for assessing the age and storage conditions of leather. A three-phase model was used for explaining the results. This consists of crystalline collagen filaments that are embedded in an amorphous matrix with an interface zone between the crystalline and amorphous regions. Solid-state ¹H NMR investigations corroborate the thermal analysis results and enhance the understanding of the ageing processes.     

Application of two-dimensional near-infrared correlation spectroscopy to protein research

The purpose of the present paper is to demonstrate the potential of generalized two-dimensional (2D) near-infrared (NIR) correlation spectroscopy in studies of hydration and structure of proteins. We describe here two examples. The first example is concerned with heat denaturation process of ovalbumin in aqueous solutions and the second one deals with isomerizations with varying pH of serum albumin. New insight has been obtained into the hydration and unfolding process of secondary structures of ovalbumin and serum albumin by studying temperature- or pH-dependent correlation patterns in 2D synchronous and asynchronous spectra. Generalized 2D NIR correlation spectroscopy emphasizes spectral features not readily observable in conventional one-dimensional spectra, enabling to extract subtle but valuable structural changes concerning with the protein denaturation.     

盐酸胍诱导叶绿素变性的THz时域光谱研究

THz时域光谱对分子构型非常敏感,能快速方便的把具有相似结构的生物分子区分开来.利用THz光谱的这一技术特点,对盐酸胍诱导叶绿素a(Chl-a)和叶绿素b(Chl-b)变性进行了研究.实验结果表明,THz光谱不仅能够鉴别变性前后的叶绿素分子,而且探测到了新的实验现象.在盐酸胍作用下,两种分子的THz吸收谱中都出现一个位于1.7 THz处的峰.通过测量几种氨基酸和盐酸胍相互作用的样品后,观察到了相同位置的峰,进而验证了这个峰是由于叶绿素的C=O键和盐酸胍的N-H键相互作用形成氧键引起的.研究结果表明,运用THz光谱技术能有效地把具有相似结构的生物分子区分开来,这项技术也是监测生物分子变性的一种有力工具.