Qing-Mei Lin

Purification, electrophoretic behavior, and kinetics of iron release of liver ferritin of Dasyatis akajei

From the liver of fish Dasyatis akajei, ferritin has been isolated by thermal denaturation and ammonium sulfate fractionation and then further purified by anion exchange chromatography and gel exclusion chromatography. The molecular weight of the liver ferritin of D. akajei (DALF) was measured to be 400 kDa by PAGE. Moreover, SDS-PAGE experimentation indicates that protein shell of DALF consists of the H and L subunits with molecular weight of 18 and 13 kDa, respectively. Using isoelectric focusing with pH ranging from 5.0 to 6.0, the ferritin purified by the PAGE exhibited three bands with different pI values in the gel slab. Diameters of the protein shell and iron core were also investigated by transmission electron microscope and determined to be 10–12 nm and 5–8 nm, respectively. A kinetic study of DALF reveals that the rate of self-regulation of the protein shell rather than the complex surface of the iron core plays an important role in forming a process for iron release with mixed orders.

Role of phosphate and kinetic characteristics of complete iron release from native pig spleen ferritin-Fe

The kinetics for complete iron release showing biphasic behavior from pig spleen ferritin-Fe (PSFF) was measured by spectrophotometry. The native core within the PSFF shell consisted of 1682 hydroxide Fe3+ and 13 phosphate molecules. Inhibition kinetics for complete iron release was measure by differential spectrophotometry in the presence of phosphate; the process was clearly divided into two phases involving a first-order reaction at an increasing rate of 46.5 Fe3+/PSFF/min on the surface of the iron core and a zero-order reaction at a decreasing rate of 6.67 Fe3+/PSFF/min inside the core. The kinetic equation [C(PSFF-Fe3+)max − C(PSFF-Fe3+) t]1/2 = Tmax −Tt gives the transition time between the two rates and represents the complex kinetic characteristics. The rate was directly accelerated twofold by a mixed reducer of dithionite and ascorbic acid. These results suggest that the channel of the PSFF shell may carry out multiple functions for iron metabolism and storage and that the phosphate strongly affects the rate of iron release.

Construction of a ferritin reactor: An efficient means for trapping various heavy metal ions in flowing seawater

An apparatus consisting of two pumps, a mixer, a ferritin reactor, and a spectrophotometer was constructed to study the ability to trap various heavy metal ions (M2+) and the dynamics of a reconstituted ferritin reactor in flowing seawater. Reconstituted pig spleen ferritin (PSFr) is assembled from apo-protein shell to form a reconstituted iron core. The main components of the PSFr are its core, which contains an Fe2+:Pi stoichiometry of 6.0±0.5, reconstituted from pig spleen apoferritin (apo PSF), Fe2+, inorganic phosphate (Pi), and O2 (0.6 atm). The Fe3+—Pi clusters within the PSFr core exhibit resistance to salt ranging from 1% to 6% NaCl. The ferritin reactor consists of PSFr and an oscillating bag. Using the reactor, M2+ ions such as Cd2+, Zn2+, Co2+, and Mn2+ are directly trapped by the ferritin. We found a 1:2±0.2 stoichiometry of the trapped M2+ to the released iron as measured by chemical analysis or atomic absorption spectrometry; nontransient elements such as Na+, K+, Ca2+, etc., were scarcely trapped by the reactor. This study provides basic conditions for establishing a ferritin reactor and a convenient means for monitoring the pollution of heavy metal ions in seawater.

Spectroelectrochemical investigation of Azotobacter vinelandii bacterial ferritin

Abstract Bacterial ferritin of Azotobacter vinelandii (AvBF) contains a function in accepting electrons from platinum electrode directly for complete iron release in the absence of a mediator. The reduction potentials of electron tunnels of −125, −310, and −370 mV for iron release are determined by direct spectroelectrochemical technique, which suggests which should be defined as midpoint potentials of electron–tunnel–heme on the surface of protein shell. A kinetic study for complete iron release by the electrode reduction at −600 mV fits the zero-order reaction law.

H2-Uptake Activity, Spectra, Reduction Potentials, and Kinetics of Iron Release on the Surface of Iron Core from Azotobacter vinelandii Bacterial Ferritin

Bacterial ferritin from Azotobacter vinelandii (AvBFo has a function in H2 uptake. The Fe3+ reduction on the surface of the iron core from AvBFo is accompanied simultaneously by H2 uptake, with a maximum activity of H2 uptake of 450 H2/AvBFo. A reduction potential of −402 mV for iron reduction on the surface of the core is found. A shift to the red the protein absorbance peaks ranging from 280 to 290 nm is observed between pH5 and 9 under 100% H2 reduction. The reduction potential for iron release becomes negative at a rate of 0.025 mV/Fe2+ released. The kinetics of iron release on the surface of the core is a first-order reaction.

Effect of pH and phosphate on trapping capacity of various heavy metal ions with ferritin reactor in flowing seawater

We describe a protein reactor consisting of native liver ferritin of Dasyatis akajei (DALF) and a dialysis bag. Our goal was to study a ferritin reactor for its capacity to trap various heavy metal ions (M2+) in flowing seawater. The reactor is sensitive and inexpensive and can be operated by nonprofessional technicians. A positive relationship between the number of trapped M2+ with the DALF reactor and its concentration in the flowing seawater was observed. Both the pH in the medium and the phosphate content within the ferritin cavity strongly affected trapping capacity. It was found that the ferritin released its phosphate compound directly with a shift in pH without the need for releasing reagent, which differs from the phosphate release characteristics of horse spleen ferritin, as previously described. This behavior evidently makes the trapping capacity with the ferritin reactor weaken, indicating that this trapping capacity is tightly connected to its phosphate compound. Our study shows that a self-regulation ability of the ferritin shell rather than its phosphate compound plays an important role in controlling the rate and capacity of trapping M2+. The ferritin reactor was constructed to monitor the contamination level of M2+ in flowing seawater. Our preliminary data along with fieldwork indicate that the DALF reactor is an analytical means for effectively monitoring the contamination level of M2+ in flowing seawater.

Effect of redox mediators on nitrogenase and hydrogenase activities in Azotobacter vinelandii.

In bioelectrochemical studies, redox mediators such as methylene blue, natural red, and thionine are used to studying the redox characteristics of enzymes in the living cell. Here we show that nitrogenase activity in Azotobacter vinelandii is completely inhibited by oxidized methylene blue (MBo) when the concentration of this mediator in the medium is increased up to 72 μM. This activity in A. vinelandii is somewhat inhibited by a coenzyme, ascorbic acid (AA). However, the nitrogenase activity within the A. vinelandii cell is unchanged even for a high concentration of oxidized natural red (NRo) alone. Interestingly, these mediators and AA do not have the capacity to inhibit the H2 uptake activity of the hydrogenase in A. vinelandii. Average active rates of 66 nM H2 evolved/mg cell protein/min from the nitrogenase and 160 nM H2-uptake/mg cell protein/min from the hydrogenase in A. vinelandii are found in aid of the activities of the enzymes for H2 evolution and for H2 uptake are compared. The activities of both enzymes in A. vinelandii are strongly inhibited by thionine having high oxidative potential. Mechanisms of various mediators acting in vivo for both enzymes in A. vinelandii are discussed.

Studies on the heme and H2-uptake reaction from Azotobacter vinelandii bacterial ferritin

Bacterial ferritin of Azotobacter vinelandii (AvBF) is directly able to pick electrons up for iron release from or transfer them for storage to a platinum electrode in the absence of mediator or other reducer. The ferritin containing the structure of heme-Co2+ in part shows weakened activity to electrode and decreases the rate of iron release greatly. A reversible reduction process of the ferritin is observed by the spectral change regularly ranging from 310 to 260 nm under mixed gases containing 98% H2 and 2% to O2. The activity of nitrogen fixation from the whole cell of A. vinelandii increases greatly by H2 reduction with potentials ranging from -397 to -425 mV vs. NHE, indicating two important roles of H2-uptake reaction of the ferritin in increasing activity of nitrogen fixation and in supplying iron to synthesize nitrogenase.