Pressure-induced Dissociation and Denaturation of Allophycocyanin at Subzero Temperatures

Abstract

The thermodynamics of assembly of the allophycocyanin hexamer was examined employing hydrostatic pressures in the range of 1 bar to 2.4 kbar and temperatures of 20 to ؊12°C, the latter made possible by the decrease of the freezing point of water under pressure. The existence of two processes, dissociation of the hexamer into dimers, (␣␤) 3 3 3 (␣␤), and dissociation of the ␣␤ dimers into monomers, (␣␤) 3 ␣ ؉ ␤ have been recognized previously by changes in the absorbance and fluorescence of the tetrapyrrolic chromophores owing to added ligands. The same changes are observed in the absence of ligands at pressures of under 2.4 kbar and temperatures down to ؊12°C. On decompression from 2.4 kbar at 0°C, appreciable hysteresis and a persistent loss of 50% in the absorbance at 653 nm is observed. It results from the conformational drift of the isolated subunits and is reduced to 10% when the highest pressure is limited to 1.6 kbar. The thermodynamic parameters of the reaction ␣ ؉ ␤ 3 ␣␤ can be determined from pressure effects on perchlorate solutions of allophycocyanin, which consist of dimers alone. Their previous knowledge permits estimation, under suitable hypotheses, of the thermodynamic parameters of the reaction 3(␣␤) 3 (␣␤) 3 from the overall pressure effects on the hexamers. Both association reactions have positive enthalpy changes, and the whole hexamer assembly is made possible by the excess entropy.Allophycocyanin is one of the phycobiliproteins of phycobilisomes, the accessory light-harvesting complexes in cyanobacteria and red algae. Allophycocyanin has been isolated as a trimeric species (␣␤) 3 (Zilinskas et al., 1978;MacColl et al., 1981) of 110 kDa to which we shall refer here as a hexamer. Conversion of (␣␤) 3 into ␣␤ dimers is obtained by the addition of 1 M perchlorate (MacColl et al., 1971;MacColl et al., 1981;Huang et al., 1987) or by lowering the pH (MacColl et al., 1980). Dissociation of ␣␤ dimers into ␣ ϩ ␤ monomers has been achieved only in the presence of urea at 8 M concentration (Erokhima and Krasnoviskii, 1974).Hydrostatic pressure has been used successfully to explore the thermodynamics and mechanism of assembly of oligomeric proteins. (Silva and Weber, 1993). In this report, we describe how the dissociation of (␣␤) 3 into ␣␤ dimers and then into ␣ ϩ ␤ monomers occurs when the solutions of the protein are subjected to hydrostatic pressures in the range of atmospheric to 3 kbar and temperatures in the range of 10 to Ϫ11°C. The decrease in the freezing point of water under pressure makes it possible to reach a temperature of Ϫ20°C at 2.4 kbar while maintaining the liquid state. In the absence of perchlorate or urea, complete dissociation of allophycocyanin takes place only at these low temperatures, and this has enabled us to show that the process of dimer dissociation by the cold results in a state very similar to that obtained by addition of a classical chemical denaturant, urea, but as a freely displaceable equilibrium. Separation of the two sequential processes of hexamer and dimer disso...

Inhibitory Activities of Proanthocyanidins from Persimmon against Oxidative Stress and Digestive Enzymes Related to Diabetes

“…Modified proteins and amino acids contribute to pathologies such as diabetes, aging, atherosclerosis, and neurodegenerative disease (24,25). Therefore, we investigated the protective effect against protein oxidation induced by free radicals, using allophycocyanin as one of the phycobiliproteins of phycobilisomes, the accessory light-harvesting complexes in cyanobacteria and red algae, as a protein donor (26). The present study suggests that proanthocyanidins from persimmon peel exert radical scavenging activities and protective effects against oxidation induced by ROS.…”

Protective Effects of Broccoli (Brassica oleracea) against Oxidative Damage in Vitro and in Vivo

“…To investigate the protective effect against protein oxidation induced by free radicals, allophycocyanin is used as one of the phycobiliproteins of phycobilisomes, the accessory light-harvesting complexes in cyanobacteria and red algae, as a protein donor (30). Modified proteins and amino acids including protein carbonyls, carboxymethyl, and carboxyethyl lysine can also be formed by the direct attack of reactive oxygen species on protein, which contributes to pathologies such as diabetes, atherosclerosis, and neurodegenerative disease (31).…”

Localization of noncovalent complexes in MALDI-preparations by CLSM

“…For the heterodimer, the absorption shifts to 620 nm and the fluorescence emission to 630 nm. However, in the absence of high concentration of chaotropic salts such as 1M NaClO 4 , their dissociation is about two orders of magnitude faster than that of the hexamer, so the heterodimer equilibrium concentration remains negligibly small compared to that of the subunits [29]. Single subunits show a weak absorption in the range of 530 nm to 650 nm and negligible fluorescence.…”

“…The concentration of the complex was adjusted to 6*10 Ϫ8 M, far above the dissociation constant of approx. 5*10 Ϫ10 M [24,29]. The fluorescence was excited at a wavelength of 647 nm and the emission detected in the range above 665 nm, identical to the parameter setting in the CLSM measurements.…”