棕色固氮菌细菌铁蛋白捕获能力、稳定性和亚基相互作用的强度

Trapping Capacities,Stability and Interaction Intensity of Subunits from Bacterial Ferritin of Azotobacter Vinelandii

选用柱层析、电泳和反相高效液相色谱（RP-HPLC）技术制备质谱纯棕色固氮菌细菌铁蛋白（Bacteri-al ferritin ofAzotobacter vinelandii,AVBF）,并采用释放铁动力学和肽质量指纹图谱（Peptide mass fingerprint-ing,PMF）技术分别鉴定AVBF。基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）和电泳技术揭示AVBF亚基之间相互作用强度、稳定性和聚合态。AVBF可直接捕获有机小分子亚甲蓝（MB）,其捕获率为15.0±2.0MB/AVBF,认为介于AVBF亚基单体之间的血红素参与捕获MB。较高浓度（40%～50%）的乙腈和丙酮均能使AVBF和鲨鱼肝铁蛋白（Liver ferritin of shark,SLF）释放不稳定亚基,但在较低浓度（20%～30%）的乙腈条件下,却需要借助来源于质谱仪的激光才能使AVBF或SLF释放不稳定亚基,并供质谱分析。AVBF亚基之间的相互作用强度明显低于SLF。铁蛋白亚基之间的相互作用强度高低与铁蛋白执行释放和储存铁的速率有关。

Bacterial ferritin with purity for the mass spectrometric analysis was prepared by the column chromatography, the electrophoresis and the RP-HPLC in Azotobacter vinelandii. Bacterial ferritin of Azotobacter vinelandii （AVBF） was further identified by both kinetics of iron release and peptide mass fingerprinting （PMF）, respectively. Moreover, the interaction intensity, stability and polymer among subunits in AVBF were further revealed by both MALDI time of flight mass spectrometry and electrophoresis. AVBF can be used to trap organic small molecules such as methylene blue （MB） and the trapping rate is approximately 15.0 ＋ 2.0 MB/AVBF, which indicates that the heine component located at the interface between monomers of the ferritn subunit participates contributes to trap MB. AVBF and liver ferritin of shark （SLF） can directly release its unstable subunits for the mass spectrometric analysis under the 40% -50% condition both acetonitrile and acetone, respectively, but both proteins can not release the subunits for the analysis under the condition of 20% -30% acetone unless both proteins absorb the laser from the mass spectrometer further. It was well known that the interaction intensity among subunits in AVBF was lower than that of SLF. The interaction intensity among protein subunits were tightly connected with the rate of iron release and storage in ferritin.