Abstract
Though human L-chain ferritin has been known to be more resistant to physical denaturation than H-type ferritin, its stability characteristics and kinetic information 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 1-1). FTN was relatively thermostable at low protein concentration (0.2 g 1-1), but it became extremely thermolabile at high protein concentration (1.3 g 1-1), i.e., more than 80% of FTN was coprecipitated within 5 rain under the same heat-induced denaturation condition. Aggregation rate constant for initial 5 rain 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 α-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, 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 GdnHCl.
| Original language | English |
|---|---|
| Pages (from-to) | 125-129 |
| Number of pages | 5 |
| Journal | Biochemical and Biophysical Research Communications |
| Volume | 289 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 2001 Nov 23 |
| Externally published | Yes |
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Keywords
- Ferritin
- N-terminal domain
- N-terminal fusion mutant
- Temperature-induced aggregation
- Thermal stability
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
Cite this
Thermal stability of human ferritin : Concentration dependence and enhanced stability of an N-terminal fusion mutant. / Kim, Sung Woo; Kim, Yang Hoon; Lee, Jeewon.
In: Biochemical and Biophysical Research Communications, Vol. 289, No. 1, 23.11.2001, p. 125-129.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Thermal stability of human ferritin
T2 - Concentration dependence and enhanced stability of an N-terminal fusion mutant
AU - Kim, Sung Woo
AU - Kim, Yang Hoon
AU - Lee, Jeewon
PY - 2001/11/23
Y1 - 2001/11/23
N2 - Though human L-chain ferritin has been known to be more resistant to physical denaturation than H-type ferritin, its stability characteristics and kinetic information 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 1-1). FTN was relatively thermostable at low protein concentration (0.2 g 1-1), but it became extremely thermolabile at high protein concentration (1.3 g 1-1), i.e., more than 80% of FTN was coprecipitated within 5 rain under the same heat-induced denaturation condition. Aggregation rate constant for initial 5 rain 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 α-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, 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 GdnHCl.
AB - Though human L-chain ferritin has been known to be more resistant to physical denaturation than H-type ferritin, its stability characteristics and kinetic information 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 1-1). FTN was relatively thermostable at low protein concentration (0.2 g 1-1), but it became extremely thermolabile at high protein concentration (1.3 g 1-1), i.e., more than 80% of FTN was coprecipitated within 5 rain under the same heat-induced denaturation condition. Aggregation rate constant for initial 5 rain 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 α-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, 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 GdnHCl.
KW - Ferritin
KW - N-terminal domain
KW - N-terminal fusion mutant
KW - Temperature-induced aggregation
KW - Thermal stability
UR - http://www.scopus.com/inward/record.url?scp=0035941124&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0035941124&partnerID=8YFLogxK
U2 - 10.1006/bbrc.2001.5931
DO - 10.1006/bbrc.2001.5931
M3 - Article
VL - 289
SP - 125
EP - 129
JO - The BMJ
JF - The BMJ
SN - 0730-6512
IS - 1
ER -