1. Transamination

2. objective Master the transamination reaction
Master the paper chromatography

3. Principle:
Transamination (or aminotransfer) is a chemical reaction between an amino acid which contains an amine (NH2) group and a keto acid, which contains a keto (=O) group. In transamination, the NH2 group on one molecule is exchanged with the =O group on the other molecule. The amino acid becomes a keto acid, and the keto acid becomes an amino acid.

4. transaminase
It catalyzes the transfer of an amino group from alanine to α-ketoglutarate, the products of this reversible transamination reaction being pyruvate and glutamate.

5. α-ketoglutaric acid（ml)
Transamination reaction
REAGENTS
measurement
Control
Liver homogenate（ml) 1
incubation
37 ℃ 10 min
Boiling 10min
Alanine solution（ml)
0.5
α-ketoglutaric acid（ml)
Phosphate buffer（ml)
mixing，37 ℃ 30 min
Boiling for 10 min

6. Paper chromatography is an analytical method technique for separating and identifying mixtures that are or can be colored.(in this experiment to separate glutamate and alanine)
Paper chromatography is performed on the filter paper. The water in the paper is the stationary phase and the organic solution is the mobile phase that move along the paper by capillary action .

7. Paper chromatography Mark the paper Assemble the paper
Apply sample multiple times (smaller is better)
Four samples:
Sample for test
Control sample
Alanine standard
Glutamate standard

9. The retention factor (Rƒ) may be defined as the ratio of the distance traveled by the substance to the distance traveled by the solvent.
Rf =
Distance of compound（r）
Distance of solvent（R）

10. α-ketoglutaric acid（ml)
Procedures:
I. Transamination reaction
REAGENTS
measurement
Control
Liver homogenate（ml) 1
incubation
37 ℃ 10 min
Boiling 10min
Alanine solution（ml)
0.5
α-ketoglutaric acid（ml)
Phosphate buffer（ml)
mixing，37 ℃ 30 min
Boiling for 10 min

11. II. Paper chromatography
1. sample application
A a starting point is marked on the paper by the pencil.
A spotter of a thin capillary tube is used to place a very small spot of the solution mixture on the paper.
Measurement (2-3 times)s
Control (2-3 times)
Alanine ( once)
Glutamate (once)

12. 4. Measurement and calculation for Rf
2. development：
the mobile phase solvent will rise slowly along the paper by capillary action.
the components of the mixture will separate.
mark the position of the solvent front.
3. detection：
Spray colorless amino acid spots with ninhydrin will produce visible purple spot.
4. Measurement and calculation for Rf

13. 【cautions:】 Make sure that the sample dot less than 0.5㎝.
After spraying of ninhydrin, dry the filter paper with the electric drier.