Methodology for Amino Acids and Proteins

Mass screening test for high lysine grains

Lysine is deficient in cereals and hence screening for high lysine grains in breeding programmes is important. Lysine can be estimated colorimetrically or with an amino acid analyzer or semi-quantitatively by paper chromatography, after hydrolyzing the proteins either with enzyme or acid. But non of the above will suit the mass screening programme. A rapid simple method id ideal for screening purposes, even if it is not a very accurate one. The dye-binding method described here has been successfully adopted at various international institutes to identify high lysine lines in barley, wheat etc.

The acidic dye acrilane orange G binds to the basic amino acids – lysine and arginine in the proteins. In high lysine lines, arginine and lysine are elevated and hence, the dye binding capacity is high. However, the general increase in protein content also increase the dye-binding capacity. To avoid the effect of protein change, the dye binding value is divided by the protein value. Hence, higher dye binding capacity per unit of protein than the normal lines identifies high lysine lines.

» Buffered Dye Solution: Dissolve 2g of acrilane orange G, 15.84g citric acid, 2.98g Na2HPO4.2H2O and 0.3g thymol in one liter of distilled water at 80°C. when measured in a spectrophotometer, this buffer solution should show an absorbance of 0.65 per mL at 580nm.

Weigh 200mg of ground and defatted whole kernel powder and transfer into a 100mL conical flask.
Add 15mL of buffered dye solution and agitate to equilibrate the dye with the reactive groups of the sample (this can be done for 5min with a blender or homogenizer. Alternatively, the solution can be kept in a standard shaker for 30min).
Filter or centrifuge the suspension.
Collect or dilute the filtrate or supernatant 200-fold with water.
Read at 480nm against the original dye solution, diluted similarly 200-fold.

Dye-binding capacity of the sample is the difference between the absorption units of the sample and the original dye. Divide this value by the total protein content of the sample determined by micro-kjeldahl method to obtain the dye binding capacity per unit protein.

1. This procedure is recommended only to compare the lysine content in progenies in a breeding programme and not to get an absolute value of lysine in individual sample. Hence, the weight (200mg), volume of dye, and dilution factor should be constant for all the samples.
2. Acrilane orange G is (otherwise called Acid Orange 12 or Crocein Orange G) 1-phenylazo-2-naphthol-6-sulphonic acid monosodium salt.

1. Mertz, E T, Jambunathan, R and Misra, P S (1975) Protein Quality, Stn. Bull No. 70, Agric Experiment Stn Purdue Univ p. 14.