Unfolding of multimeric proteins in presence of denaturants. A case study of helianthinin from Helianthus annuus L.

Helianthinin (11S), a multisubunit protein from Sunflower Seeds (Helianthus annuus L.) dissociates to its monomer (2S) through a trimeric (7S) intermediate as a function of guanidine hydrochloride (GuHCl) and guanidine thiocyanate (GuHSCN) concentration. Measurements of viscosity, velocity sedimentation patterns and spectroscopic parameters of the protein in presence of these denaturants both at equilibrium and as a function of time clearly suggest that the dissociation, unfolding and aggregation of this multimeric protein occur sequentially. The unfolding of the protein in guanidinium salts has two transitions with the first transition occurring between native to intermediate and the second transition occurring between intermediate state and unfolded state. The midpoint concentration for the major transition is 1.75 M for GuHCl and 0.8 M for GuHSCN. Evaluation of this data suggests that during the process of denaturation the simultaneous unfolding of acidic and basic subunits of the protein takes place. At intermediate concentrations of denaturant namely 1.6 M of GuHCl or 1.1 M of GuHSCN the aggregation of the protein was found to be maximum. The results suggests the possibility of a mechanism for the dissociation, denaturation and unfolding of multimeric proteins in presence of the chemical denaturants.

Thermodynamics of interaction of caffeic acid and quinic acid with multisubunit proteins.

Helianthinin is a multisubunit protein from Sunflower seeds. Caffeic acid (CA) and quinic acid (QA) are intrinsic ligands present in sunflower seeds. The mechanism of interaction of these ligands with multisubunit proteins is limited. The present study enables one to understand the mechanism of the interaction of these ligands with the protein helianthinin. From this study, it is shown that CA has two classes of binding sites on helianthinin. The high-affinity class of sites total six from 60+/-10 for both high-affinity and low-affinity sites. Tryptophan, tyrosine and lysine residues of the protein are mainly involved in the interaction with CA. The temperature dependence of the binding in the range 10-45 degrees C can be clearly described by an enthalpy-entropy compensation effect at the low-affinity class of sites, while it is described by positive DeltaC(p)(o) at the high-affinity class of sites. This positive DeltaC(p)(o) has a contribution to the protein stability. The binding strength of CA also has a positive cooperativity at higher protein concentration. QA has two classes of binding sites on the protein based on the strength of the interaction. The interaction of QA with the protein is predominantly described by positive DeltaC(p)(o) for both classes of affinity. This suggests predominance of ionic/hydrogen bonding in the interaction process. Differential scanning calorimetric measurements reveal that the binding of both CA and QA induces destabilisation of the subunit-subunit interaction. Human methaemoglobin (mHb) has two binding sites on the molecule for CA. Both CA and QA decrease the stability of mHb, as indicated by decreased T(m). This destabilisation is also accompanied by dissociation to the monomers with concomitant conformational changes.

Interaction of 5,7-dihydroxy-4'-methoxyflavone with a multisubunit protein, carmin: thermodynamics and kinetics of interaction.

Acacetin (5,7-dihydroxy-4'-methoxy flavone) is a flavone intrinsically present in the seeds of Carthamus tinctorius. Carmin is a multimeric, high molecular weight protein from the seeds of Carthamus tinctorius. The association constant of interaction of acacetin and carmin is maximum at 37.2 degrees C with a value of (3.96 +/- 0.61) x 10(4) M-1 as measured by fluorescence quenching. Acacetin has at least two binding sites on carmin. The interaction follows pseudo-first-order kinetics with a reaction rate constant of 3.4 +/- 0.4 s-1. The titration calorimetric data suggest that binding sites for acacetin and its structural analogue, biochanin A, are conserved. The interaction does not affect the association-dissociation equilibrium of the protein. Also, the binding does not induce any significant conformational changes in the protein as monitored by circular dichroic spectra. Biochanin A (5, 7-dihydroxy-4'-methoxyisoflavone), a structural analogue, interacts with carmin with an association constant of (9.33 +/- 1.44) x 10(4) M-1 at 36.9 degrees C. This indicates that the stereochemistry of the ligand plays an important role in the binding process of flavone to protein. Interaction studies of chemically modified lysyl and tryptophanyl groups separately, and lysyl and tryptophanyl groups sequentially, in the protein carmin with the ligands reveal the involvement of tryptophanyl residues in the binding process and show that it is predominantly an entropically driven hydrophobic interaction.

Iodine metabolism in response to goitrogen induced altered thyroid status under conditions of moderate and high intake of iodine.

Metabolic experiments in rats were undertaken to relate excretory pattern of iodine and thiocyanate, with thyroid weight and the circulating levels of thyroxine, in response to moderate and high intake of iodine and under conditions of goitrogen induced altered thyroid status. On a moderate intake of iodine (by depriving diet of KI) 25 mg of thiocyanate or substitution of 1/3rd proportion of casein based diet with dry cabbage, could significantly reduce plasma thyroxine level by 60 days. Neither body weight nor the weights of liver, kidney, heart or spleen were affected due to exposure to goitrogens. A significant increase in thyroid weight as well as higher excretion of iodine and thiocyanate were evident in goitrogen-fed rats. Presence of high amounts of KI, to a certain extent, offered protection from adverse effects of the goitrogens. Semi quantitative assessment of thyroid, indicated hypofunctioning of thyroid with follicular hyperplasia in thiocyanate fed rats. These alterations were of moderate degree in response to cabbage feeding. These results emphasize that, moderate intake of iodine, adequate to meet iodine requirement, may not ensure normal functioning of thyroid in the presence of goitrogens.