Thermal stability of human ferritin: Concentration dependence and enhanced stability of an N-terminal fusion mutant

Though human L-chain ferritin has been known to be more resistant to physical denaturation than H-type ferritin, its stability characteristics and kinetic in-formation have not been reported in detail. Overexpressed recombinant ferritin (FTN) in Escherichia coli formed inclusion bodies through noncovalent molecular interaction and easily dissolved with regaining the iron-uptake activity by a simple pH-shift process at high protein concentration (> 600 mg l(-1)). FTN was relatively thermostable at low protein concentration (0.2 g l(-1)) but it became extremely thermolabile at high protein concentration (1.3 g l(-1)), i.e., more than 80% of FTN was coprecipitated within 5 min under the same heat-induced denaturation condition. Aggregation rate constant for initial 5 min at high protein concentration was 6.04 X 10(-3) s(-1) for FTN. Surprisingly, glucagon . ferritin mutant (GFTN), consisting of an N-terminus fusion partner, human glucagon (29-residue a-helical peptide), showed significantly enhanced thermal stability even at high protein concentration. That is, in spite of 40-min heat treatment, more than 50% of GFTN the still remained soluble with maintaining the same functional properties. The aggregation rate constants were 2.75 X 10(-4) and 2.80 X 10(-4) s(-1) at low and high concentration, respectively, for GFTN. These results suggest a critical participation of the N-terminal domain of ferritin in the temperature-induced aggregation pathway. Presumably, partially denatured amino terminus of FTN is involved in nonspecific molecular interaction resulting in the off-pathway aggregation. It is notable that the purified GFTN showed the same molar capacity of iron (Fe+3) storage as standard ferritin. From the analysis of fluorescence emission spectrum, the physical stability of GFTN was also very comparable to that of standard ferritin under the various denaturation conditions induced by GdnHCI. (C) 2001 Academic Press.