Determination of the total amount of free amino acids in plants
Last Update: 2021-01-08
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amino acids can react with hydrated tritones, resulting in blue-purple compounds, the depth of its color is directly related to the content of free amino acids.
; capacity bottle 25ml or 50ml 3; funnel (6 cm 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 test tube port); absorbent paper; mirror paper moderate amount; electric stove; water bath pot (including wire basket).
1.3% thyone reagents
said 3g tritones with 95% ethanol dissolved into 100ml capacity bottles, stored in brown bottles. 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 scale with 10% isopropyl alcohol in 100 ml capacity bottles, mixed, i.e. standard amino acid storage liquid of 1mmol/L, stored in a 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;
, the operation steps
1. The production of the standard curve take 18 test tubes of 20 ml scale, add each reagent according to Table 1. After adding the reagent mixed, in a water bath at 100 degrees C
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 tube as the reference), take amino acid concentration as horizontal coordinates, light density as ordinate, draw standard curves, and find straight-line equations.
2. Sample extraction Select representative plant leaves (or other
), wash and dry, cut and mix well, quickly weigh 0.10 to 0.20g (depending on the amino acid content), a total of 3 servings, respectively, added to the 20ml scale test tube, and then Add distilled water 10 ml plug (or tie plastic film), put 20min in boiling water bath to extract free amino acids, then remove in tap water cooling, filter the upper liquid into a 25ml capacity bottle, and then add 5 ml of distilled water to the test tube, heat 10min on the boiling water bath,
and rinse the residue repeatedly, and finally set the scale and shake well.
3. Sample determination 4 clean
test tubes, three of which were added with 0.5 ml of extract and the other with 0.5 ml of distilled water, and then added NaCN buffer and hydration to each of the four test tubes mentioned above
The three ketones are 0.5 ml each, after the reagent is added to the plug, heated on the boiling water bath 12min, cooled, then added 5 ml 95% ethanol, shake well, with blank as a test, at a wavelength of 570nm to measure its light density.
the concentration of amino acids in the extract can be calculated by checking the standard curve of light density (or calculated by regression equation).
4. Calculating the value of
free amino acid content
: the value calculated by the linear equation, i.e. the number of micromolars of amino acids in the 0.5 ml sample( smol);
V: the diluted total product (ml) of the sample extract;
W: sample weight (g).
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