Methodology for Amino Acids and Proteins

Estimation of tryptophan



Cereals like maize and sorghum are deficient in tryptophan in addition to lysine. Methods to determine these essential amino acids are useful to identify nutritionally superior types.


Principle
 The indole ring of tryptophan gives an orange-red color with ferric chloride under strongly acidic condition. The color intensity is measured at 545nm.




Materials
» Papain Solution: Dissolve 400mg technical grade papain in 100mL 0.1N sodium acetate buffer pH 7.0. prepare the solution fresh every day.
» Reagent A: Dissolve 135mg FeCl3.6H2O in 0.25mL water and dilute to 500mL with glacial acetic acid containing 2% acetic anhydride.
» Reagent B: 30N H2SO4.
» Reagent C: Mix equal volumes of reagent A and B about one hour before use.
» Standard Tryptophan: Dissolve 5mg Tryptophan in 100mL water (50mg/mL)




Procedure
1.
Weigh 100mg of air-dried, powdered and defatted grain sample into a small test tube.
2.
Add 5mL papain solution, shake well and close the tube.
3.
Incubate at 65°C overnight.
4.
Cool the digest to room temperature, centrifuge and collect the supernatant.
5.
To one mL supernatant add 4mL reagent C.
6.
Mix in a vortex mixture and incubate at 65°C for 15min.
7.
Cool to room temperature and read the orange-red color at 545nm.
8.
Set a blank with 5mL papain alone and repeat steps 3 to 7.
9.
Pipette out 0, 0.2, 0.4, 0.6, 0.8 and 1mL standard tryptophan and make up to 1mL with water. Develop color following the steps 5 to 7.
 

Calculation
Draw a standard curve. Subtract the absorbance value of the blank from that of the sample and calculate tryptophan content from the graph.
Tryptophan in grain sample   =
Tryptophan value from the graph in mg x 6.4
Percent N in the sample
                                                        =      g/16 gN.


References
1. Mertz, E T, Jambunathan, R and Misra, P S (1975) In: Protein Quality Agricultural Research Stn. Bull No. 7 Purdue Univ  USA p. 9.
2. Theymoli Balasubramanian and Sadasivam, S (1987) Plant Foods Hum Nutr 37 41.