Based on the three-state model of protein denaturation-renaturation, an equation describing the effect of protein concentration in denaturant solution on renaturation yield of denatured protein was presented. Through this equation two important parameters, the number n of denatured protein molecules included in an aggregate and the apparent aggregation equilibrium constant K for protein denaturation from native state to aggregate state, could be simultaneously derived. By using lysozyme as model protein, this equation was tested with the renaturation process of three kinds of denatured lysozyme molecules in urea and guanidine hydrochloride solutions, and it was found that the renaturation results of denatured lysozymes could be described well by this equation, and these denatured lysozyme molecules had a tendency to form a bi-molecular intermediate aggregate in both solutions. This result was further confirmed with their SDS-PAGE and high-performance size-exclusion chromatography.

Key words: lysozyme, aggregate, denaturation-renaturation, three-state model