Unfolding-refolding behaviour of chicken egg white ovomucoid and its correlation with the three domain structure of the protein.
The urea and heat-induced unfolding-refolding behaviours of chicken egg white ovomucoid and its four fragments representing domains I, II + III, I + II and III were systematically investigated in 0.06 M sodium phosphate buffer (pH 7.0) by difference spectral measurements. The effect of temperature on ovomucoid and its fragments was also studied in 0.05 M sodium acetate buffer (pH 5.0) and in presence of 2 M urea at pH 7.0. Intrinsic viscosity data showed that ovomucoid and its different fragments did not lose any significant amount of their structure under mild acidic conditions (pH 4.6). Difference spectral results showed extensive disruption of the native structure by urea or temperature. Isothermal transitions showed single-step for domain I, domain I + II and domain III, and two-step having one stable intermediate, for ovomucoid and its fragment representing domain II + III. However, the presence of intermediate was not detected when the transitions were studied with temperature at pH 7.0. Strikingly, the single-step thermal transitions of ovomucoid and its fragment representing domain II + III, became two-step when measured either at pH 5.0 or in presence of 2 M urea at pH 7.0. Analysis of the equilibrium data on urea and heat denaturation showed that the second transition observed with ovomucoid or domain II + III represent the unfolding of domain III. The kinetic results of ovomucoid and its fragments indicate that the protein unfolds with three kinetic phases. A comparison of three rate constants for the unfolding of intact ovomucoid with that of its various fragments revealed that domain I, II and III of the protein correspond to the three kinetic phases having rate constants 0.456, 0.120 and 0.054 min-1, respectively. These data have led us to conclude: (i) the unusual stability of ovomucoid towards various denaturants, including temperature, is due to its domain III, (ii) initiation of the folding of the ovomucoid molecule starts from its NH2-terminal region which probably provides the nucleation site for the formation of the subsequent structure and (iii) domains I and II have greater mutual recognition between them as compared to the recognition either of them have with domain III.
Das
B K
BK
Department of Biochemistry, School of Life Sciences, North-Eastern Hill University, Shillong, India.
Agarwal
S K
SK
Khan
M Y
MY
D000818:Animals; D002645:Chickens; D004529:Egg White; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010059:Ovomucin / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
D006146:Guanidines; D010059:Ovomucin; D014508:Urea; D019791:Guanidine