Fluorescence properties and stability of dioxygen--binding functional units from the Rapana thomasiana hemocyanin subunit RHSS2

Molecular aggregates of the Rapana thomasiana hemocyanin are composed of two structural subunits, RHSS1 and RHSS2, each of which contains eight functional units (FUs) reversibly binding dioxygen. Multiunit fragments and individual 50-60 kDa FUs from RHSS2 were isolated and characterized by electron and fluorescence spectroscopy. The units have similar fluorescence parameters demonstrating that the tryptophyl side chains are located in the hydrophobic core of the globular folded regions. The copper-dioxygen system at the binuclear active site stabilizes considerably the native protein structure and quenches the indole emission. The removal of this system decreased the 'melting points' drastically Tm by 13-20 degrees C and increased 2-4 times the fluorescence quantum yields. The individual FUs differ considerably in their thermostability. The activation energy for the thermal deactivation of the excited tryptophyl residues of the apo-FUs is lower compared to that of the whole molluscan apo-Hcs.     

Rapana thomasiana hemocyanin (RtH): comparison of the two isoforms, RtH1 and RtH2, at 19A and 16A resolution

Three-dimensional (3D) reconstructions of the two 8.4 MDa Rapana thomasiana hemocyanin isoforms, RtH1 and RtH2, have been obtained by cryoelectron microscopy of molecules embedded in vitreous ice and single particle image processing. The final 3D structures of the RtH1 and RtH2 didecamers at 19 A and 16 A resolution, respectively, are very similar to earlier reconstructions of gastropodan hemocyanins, revealing structural features such as the obliquely oriented subunits, the five- and two-fold symmetrical axes. Three new interactions are defined; two of them connecting the arch and the wall while the third is formed between the collar and the wall. The collar-wall connection and one of the arch-wall connections are positioned between two individual subunit dimers, while the second arch-wall connection is located between two subunits within the subunit dimer. All three interactions establish connections to the first tier of the wall. Furthermore, for each interaction we have allocated two first tier functional units most likely involved in forming the connections.     

Spectroscopic Properties and Conformational Stability of Concholepas concholepas Hemocyanin

The structure in solution and conformational stability of the hemocyanin from the Chilean gastropod mollusk Concholepas concholepas (CCH) and its structural subunits, CCH-A and CCH-B, were studied using fluorescence spectroscopy and differential scanning calorimetry (DSC). The fluorescence properties of the oxygenated and apo-form (copper-deprived) of the didecamer and its subunits were characterized. Besides tryptophan residues buried in the hydrophobic interior of the protein molecule also exposed fluorophores determine the fluorescence emission of the oxy- and apo-forms of the investigated hemocyanins. The copper-dioxygen system at the binuclear active site quenches the tryptophan emission of the oxy-forms of CCH and its subunits. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophan residues in the respective apo-forms. Time-resolved fluorescence measurements show that the oxygenated and copper-deprived forms of the CCH and its subunits exist in different conformations. The thermal denaturation of the hemocyanin is an irreversible process, under kinetic control. A successive annealing procedure was applied to obtain the experimental deconvolution of the irreversible thermal transitions. Arrhenius equation parameter for the two-state irreversible model of the thermal denaturation of oxy-CCH at pH 7.2 was estimated. Both factors, oligomerization and the copper-dioxygen system at the active site, are important for stabilizing the structure of the hemocyanin molecule.     

Rosmarinic acid-conjugated hemocyanins: synthesis and stability

Journal of Thermal Analysis and Calorimetry - Two oxygen-transporting proteins known for their immunostimulating and anticancer properties, namely hemocyanin from Helix lucorum (HlH) and hemocyanin...     

Structural,Thermal, and Storage Stability of Rapana Thomasiana Hemocyanin in the Presence of Cholinium-Amino Acid-Based Ionic Liquids

Novel biocompatible compounds that stabilize proteins in solution are in demand for biomedical and/or biotechnological applications. Here, we evaluated the effect of six ionic liquids, containing mono- or dicholinium [Chol]_1or2 cation and anions of charged amino acids such as lysine [Lys], arginine [Arg], aspartic acid [Asp], or glutamic acid [Glu], on the structure, thermal, and storage stability of the Rapana thomasiana hemocyanin (RtH). RtH is a protein with huge biomedicinal potential due to its therapeutic, drug carrier, and adjuvant properties. Overall, the ionic liquids (ILs) induce changes in the secondary structure of RtH. However, the structure near the Cu-active site seems unaltered and the oxygen-binding capacity of the protein is preserved. The ILs showed weak antibacterial activity when tested against three Gram-negative and three Gram-positive bacterial strains. On the contrary, [Chol][Arg] and [Chol][Lys] exhibited high anti-biofilm activity against E. coli 25213 and S. aureus 29213 strains. In addition, the two ILs were able to protect RtH from chemical and microbiological degradation. Maintained or enhanced thermal stability of RtH was observed in the presence of all ILs tested, except for RtH-[Chol]₂[Glu].     

Investigation on chemical cross-linked collagen phosphoric acid hydrolysates with cyanuric chloride by differential scanning calorimetry

The process of cross-linking of collagen phosphoric acid hydrolysates (CH) with cyanuric chloride (CY) was studied by the increase in the denaturation temperature using differential scanning calorimetry (DSC). This measurement gave indications concerning the efficiency of the treatment, i.e., the extent of cross-linking of the collagen hydrolysates. The optimal conditions for cross-linking were determined: CH/CY in a ratio 1:1, reaction time 1 h at temperature 50 °C. At these conditions cross-linked structural units with higher thermal stability were formed.     

Rapana thomasiana hemocyanin (RtH): dissociation and reassociation behavior of two isoforms, RtH1 and RtH2

Rapana thomasiana hemocyanin (RtH) is a mixture of two hemocyanin isoforms, termed RtH1 and RtH2. The two subunit types, purified by ion exchange chromatography, were used for macromolecular reassociation studies. In vitro reassociation was achieved with Tris-saline stabilizing buffer at pH 7.4, containing 100mM calcium and magnesium chloride at 4 degrees C. The relatively slow progress of reassociation was monitored, and the different oligomeric forms of RtH1 and RtH2 were studied by transmission electron microscopy, using samples negatively stained with 1% (w/v) uranyl acetate or 5% (w/v) ammonium molybdate containing 1% (w/v) trehalose at pH 7.0. The two subunits reassociate to produce characteristic didecamers, oligomeric and polymeric forms depending on the dissociated material and the reassociation conditions (i.e. divalent ion concentration, duration). In contrast to the didecamers of the freshly isolated RtH preparations, RtH1 and RtH2 show after 2 weeks' reassociation a clear tendency to generate multidecameric structures. The behavior of the native RtH1 and RtH2 during reassociation in the presence of 100mM calcium and magnesium chloride corresponds to the reported common oligomerization characteristics of KLH1/HtH1 and KLH2/HtH2, respectively. It is important to note that during the reassociation of the RtH isoforms: (I) no smaller diameter tubular polymers (ca. 25-27nm) were formed from the subunits as well as from the decamers; (II) multidecamers with one or more 'nucleating' didecamers were detected in addition to the multidecamers, composed of didecamers with associated decamers at one or both ends.     

Thermal stability and secondary structure of feruloylated Rapana thomasiana hemocyanin

Journal of Thermal Analysis and Calorimetry - This is the first report on the conjugation of a hemocyanin from Rapana thomasiana (RtH) with ferulic acid, which itself exhibits an immunostimulatory...