Team:Korea U Seoul/Project/Protocols

Method

The overall reaction for precipitation is

Ca2+ + HCO3 - → CaCO3 + H+

According to that equation, CaCO3 generated the more the pH will be lowered.

The effect of NA and R on the rate of precipitation of CaCO3 was determined by recording the decrease in pH of a 20mM Tris HCl pH8.0 buffer solution at 10°C

Reagents

• 20mM Tris-HCl buffer, pH 8.0.
• 20mM Tris-HCl buffer, pH 8.0 + 1M Imidazole.
• 1M NaHCO3.
• 1M CaCl2.
  
  
  

Enzyme

Using Protino® Ni-NTA Columns for His-tag protein purification, purify the enzymes.

These enzymes is diluted in 20mM Tris-HCl buffer, pH 8.0 + 10mM Immidazole at 4°C.

Procedure

• Blank Determination
Add 0.5 ml of 1M CaCl2 to a 15 ml tube cotaining 0.5 ml of 1M NaHCO3 and 9 ml of 20mM Tris-HCl buffer. Immediately start a stop watch and record the time and pH up to 20 minutes.
• Enzyme Determination
Add freshly diluted enzyme to a 15 ml tube cotaining 0.5 ml of 1M NaHCO3 and 9 ml of 20mM Tris-HCl buffer. Add Add 0.5 ml of 1M CaCl2. Immediately start a stop watch and record the time and pH up to 20 minutes.

Method

The electrometric method of Wilbur and Anderson (1948) in which the time required (in seconds) for a saturated CO2 solution to lower the pH of 0.012 M Tris⋅HCl buffer from 8.3 to 6.3 at 0°C is determined. The time without enzyme is recorded at T0; with enzyme, T.

A unit of activity = (2×(T_0-T))/T

Reagents

0.02 M Tris⋅HCl buffer, pH 8.0. Store in an ice bath at 0-4°C before and during use.
Carbon dioxide saturated water. Bubble CO2 gas through 200 ml ice cold water for 30 minutes prior to assay. During saturation process, store water at 0-4°C in an ice bath.

Enzyme

Using Protino® Ni-NTA Columns for His-tag protein purification, purify the enzymes.
These enzymes is diluted in 20mM Tris-HCl buffer, pH 8.0 + 10mM Immidazole at 4°C.

Procedure

• Blank Determination
Add 6.0 ml of 20 mM Tris⋅HCl buffer, pH 8.0 to a 15ml tube. Maintain temperature at 0-4°C and record pH. Withdraw in a 5 ml syringe, 4 ml of chilled CO2 saturated water and add to Tris buffer. Immediately start a stop watch and record the time required for the pH to drop from 8.3 to 6.3. Record this time as T0.


• Enzyme Determination
Add 6.0 ml of 20 mM Tris⋅HCl buffer, pH 8.0 to a 15ml tube. Maintain temperature at 0-4°C and record pH. Add 0.1 ml of freshly diluted enzyme. Quickly add 4 ml of CO2 saturated water and record the time required for the pH to drop from 8.3 to 6.3. Record this time as T.