금속 친화성 액 이상분계 시스템에서 Phsphoprotein분배 및 분배예측

Phosphoprotein Partitioning in Metal-Affinity Aqueous Two-Phase Systems and Prediction of Partitioning Behavior

Fe(III) IDA -PEG/ dextran 액 이상 분계 시스템에서 phosphoprotein의 분배 예측을 위한 모델식이 유도되었다. 본 모델식에는 단백칠 분배에 수소이온과 hydroxide 이온의 저해효과가 포함되어 었다. 표변에 각각 1,27~의 phosphoryl 그 룹을 갖고 있는 ovalbumin의 AI, A2, 성분을 정제한 후 단백질 분배 실험을 수행하였으며 실 험결과와 모텔식에의한 예측이 잘 얼치하는 것 을 확인하였다. Fe(III)IDA-PEG와 단백질 표면 p phosphoryl group과의 결합상수는 PEG 상과 dextran상에 셔 각각 $6.1{\times}, 10^3M^{-1} and 2.3{\times}10^4M^{-1}$이었으며,이것은 Cu(II)IDA-PEG와 단백질 표면 histidine과의 결합상수 보다 3-5배 높은 값이 였 다.

A mathematical model has been derived and used to describe phosphoprotein partitioning in Fe(III) IDA-PEG/dextran two-phase systems. This model includes the inhibitory effects of hydrogen and hydroxyl ion concentrations on protein partitioning. For aqueous two-phase partitioning experiments, the Al and A2 subcomponents of ovalbumin carrying two and one surface phosphoryl group(s) were purified using an immobilized metal ion affinity chromatography (IMAC). The ratio of partition coefficients in the presence and absence of Fe(III)IDA-PEG, K/Ko, increased in the pH range of 3.0 to 5.0 due to deprotonation of the second oxygen of the phosphoryl group, and above pH 5.0 declined steeply by the inhibitory binding of hydroxyl ions to the metal ion. This partitioning behavior was well described by the mathematical model. The binding constants for formation of the complex between the phosphoryl group and the Fe(III)IDA-PEG were found to be $6.1{\times}10^3M^{-1} and 2.3{\times}10^4M^{-1}$ in the top and bottom phases, respectively. These values are 3-5 times those for interaction of Cu(II)IDA-PEG with a single surface-accessible histidine.