Determination of the total amount of free amino acids in plant tissue

to determine the content of free
amino acids
in plants is of great significance for studying the changes of nitrogen metabolism in plants under different conditions and during different growth and development periods, the absorption, transportation, assulation and nutritional status of plants.
principle
the amino acids of free amino acids can react with hydratedtritones to produce blue-purple compounds whose color is directly related to the content of free amino acids.

equipmentternometer; electronic
balance
; capacity bottles 25ml or 50ml 3; funnel (6cm diameter) 3, filter paper suitable; 20ml scale
test tube

7; pipe pipe 0.5ml 3, 5ml 1; test tube holder; glass rod; ear-sucking ball; scissors; pipe pipe rack; rubber band, plastic film (sealed tube port); water-absorbing paper; mirror paper moderate amount; electric stove; water bath pot (including wire basket).

1.3% thyone reagents
called 3gthone dissolved with 95% ethanol into a 100ml capacity bottle and stored in a brown bottle. This reagent should be placed in a cold place, should not be put for a long time, the use period of about 10 days.
2. Cyanate buffer (prepared as follows):
(1) NaCN storage fluid 0.01mol/L (490mg/L).
(2) acetate buffer: said 360g sodium acetate (containing three molecule crystalline water) dissolved in about 300 ml of ammonia-free distilled water, plus 66.67 ml of ice acetic acid and diluted to 1L with ammonia-free distilled water.
the solution (1) 20 ml, with the solution (2) to 1L.
3. Standard amino acids precisely named 13.1 mg (or α-alanine 8.9 mg) dried at 80 degrees C are dissolved in 10% isopropyl alcohol and diluted to the scale with 100 ml of capacity bottles, mixed, i.e. 1mmol/L standard amino acid storage liquid, stored in the refrigerator. In order to prepare the working fluid, it is desirable to mix the storage fluid with the same amount of ammonia-free distilled water at a concentration of 0.5 mmol/L, i.e. 1 ml containing the amino acid 0.5 μmol, or amino nitrogen 7 μg.
4. 95% ethanol; 5. isopropyl alcohol (analytical pure).
the "Procedure"
1. The production of the standard curve take 18 test tubes of 20 ml scale, and add each reagent under table 15-1. After adding the reagent mixed, in a water bath at 100 degrees C
heat
12min (sealed when heated), remove in cold water to cool quickly, immediately add 5 ml of 95% ethanol in each tube, plug the plug, shake the test tube, so that the red product formed during heating is oxidized by oxygen in the air and faded, at this time the solution is blue-purple. Measure its light density at 570nm wavelengths (with blank tubes as the reference), take amino acid concentration as horizontal coordinates, light density as ordinate, draw standard curves

, find out the linear equation
Depending on the acid content), a total of 3 parts, respectively, add 20 ml scale test tube, add distilled water 10 ml plug (or tie plastic film), put 20min in the boiling water bath to extract free amino acids, then remove in tap water cooling, liquid filter into 25ml capacity bottle, and then add 5 ml of distilled water to the test tube, heating 10min on the boiling water bath,
filter
and rinse the residue repeatedly, and finally set the scale and shake well.
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