Characterization of Stable Conformational States in Urea-Induced Transition in Ovomucoid

Abstract

The urea-induced transition of ovomucoid, which was studied by viscosity, difference-spectral and fluorescence measurements in 0.06 M phosphate buffer, pH 7.0, has been shown to occur in two steps involving at least three conformational states, that is, the native, the intermediate and the fully denatured states. Equilibrium and kinetic studies showed that the two transitions were completely reversible. These states have been characterized by solvent perturbation of the absorption and fluorescence spectra of ovomucoid as well as by viscosity measurements. Perturbation results with ethylene glycol and propylene glycol suggested an exposure of about three phenolic residues in the native ovomucoid. As judged by the intrinsic viscosity (5.3 ml/g), the native protein seems to exist in a conformation more expanded than that of a typical globular protein. In the intermediate state, which obtains at 6 M urea, the intrinsic viscosity of ovomucoid was measured to be 6.44 ml/g. As a result of this small but significant unfolding, the exposure of tyrosyl residues was increased ; probably an additional phenolic group became exposed. The fully denatured state occurs near and above 9 M urea. As measured by intrinsic viscosity (7.2 ml/g), the effective hydrodynamic volume increased by 12 % upon transition of the intermediate state to the denatured state. Solvent perturbation data indicated exposure of nearly all of the tyrosine residues of ovomucoid in the denatured state. These along with the intrinsic viscosity of ovomucoid (14.3 ml/g) in 9 M urea containing 0.1 M 2-mercaptoethanol have shown that the protein probably loses almost all the elements of its native structure in 9 M urea. Analysis of the preliminary kinetic results indicated that the refolding is faster than the unfolding process for both the transitions; native + intermediate s denatured.The final step in protein biosynthesis is the folding of polypeptide chain into a native and functional conformation. Useful information about the mechanism of the overall process of protein folding can be obtained by characterizing the different conformational states involved in the folding-unfolding process [l]

Thermodynamics of unfolding for turkey ovomucoid third domain: Thermal and chemical denaturation

“…OMTKY3 is highly soluble and stable over a wide range of pH and temperature (Ardelt & Laskowski, 1991), and its structure is well characterized both in the solid state and in solution (Read et al, 1983;Read & James, 1986;Robertson et al, 1988). Whole ovomucoid and its third domains have proven to be propitious for biophysical investigations (Waheed et al, 1977;Baig et al, 1978;Watanabe et al, 1981;Matsudaet al, 1981a,b;Griko & Privalov, 1984;Hildebrandt et al, 1988;Das et al, 1990Das et al, , 1991Ardelt & Laskowski, 1991). The present studies, while focused on determination of AC, for OMTKY3, have also provided opportunities to test and develop methods for characterization of the thermodynamics of unfolding.…”

Protein proteinase inhibitors from avian egg whites

“…Tryptophan is absent in ovomucoid. About half of the tyrosyl residues of the protein are exposed under native conditions, as shown by solvent perturbation difference spectroscopy [17]. Ovomucoid is rich in istructure (46%) compared with h-structure (26%), as studied by circular dichroism [18].…”

“…NMR studies of different ovomucoid third domains suggest that they behave as a rigid, native, globular protein with high melting temperatures [21]. Denaturation studies of ovomucoid and its isolated domains suggest that these domains fold independently into a compact shape with a hydrophobic core [17,[21][22][23][24]. The third domains of silver pheasant, Japanese quail and turkey ovomucoids (virgin and modified) have been isomorphically crystallized in the monoclinic space group C2 with cell dimensions of a=4.429 nm, b= 2.115 nm, c= 4.405 nm and i= 107° [25][26][27].…”

High Pressure Bioscience

“…32.5c). Though the unfolding of OVM that proceeds in two distinctive steps with a intermediary formation in between them has been shown using urea and guanidine hydrochloride (Waheed et al 1977;Mustafa et al 2011), studies on pressure axis, which can give the structural as well as thermodynamic information on semi-stable high energy conformers of this protein located higher on the folding funnel , have not been carried out. In these proteins, except for chymotrypsino- 11 % of the total protein content and is the most potent allergen in egg white.…”