The Research On Denaturation And Renaturation Of Human Placental Alkaline Phosphatase

The changes of conformation and activity of denatured and renatured human placental alkaline phosphatase(HPAP, E. C 3. 1. 3. 1) in guanidine solutions of different concentrations are studied by spectrophotometry and fluorometry. HPAP refolding mechanism is visited. The result are showed that.: (1)A guanidine concentrations lower than <0. 5mol/L, fluorescence emission intensity decreases without any red shift of emission maximum, 340nm, and this is accompanied by a marked increase in HPAP activity. A guanidine concentrations higher than 0. 5mol/L, increase in fluorescence emission intensity occurs and the enzyme is gradually inactivated. When the guanidine concentration is 1. 5mol/L, fluorescence emission maximum shifts red and the enzyme activity is rapidly decreased. At 3. 0 mol/L guanidine, fluorescence emission wavelength is red shift to maximum,365nm, and the enzyme is completely inactivated. At 4.0mol/L guanidine, fluorescence emission intensity continues to increase. Only to 5.0mol/L guanidine it stops to change. Changes of the fluorescence emission spectra and activity of denaturation-equilibrated state HPAP in guanidine solution of different concentration occurred in two stages. All indicated that a three-state unfold mechanism with an intermadiate state is involved in the denaturation process. (2)The guanidine denaturation HPAP are refolded with dilution method and atmosphere oxiation method . It's the most fitted renaturation condition is such as: A guanidine denaturation concentration 3. Omol/L, a guanidine dilution concentration 0. 5mol/L, renaturation temperature 4C, renaturation buffer pH 9, renaturation time 24 hours. The enzyme refolding rate is 27 % with these renaturation conditions, and fluorescence emission wavelength is blue shift from 365nm to 358nm. (3)After mercaptoethanol is added in the refolding process, the denaturation intermadiate state HPAP can still renaturation completely; but the renaturation rates of denaturated state have been improved obviously. Its refolding rate raises from 27% to 68. 5%, and fluorescence emission wavelength shifts blue from 358nm to 347. 5nm. The results indicate that: HPAP have three states in the denaturation process, and they are national state, denaturation intermadiated state and denatured state; The denaturation intermadiated state HPAP can been completely refolded . The denatured state are partial refolded and their refolding rates are related to renaturation conditions. How do we make the denatured state HPAP renaturation completely? It need still inquire about its the most fitted renaturation conditions.