6 Estimation of lysine
 

 

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The protein in the grain sample is hydrolysed with a proteolytic enzyme, papain. The alpha-amino groups of the derived amino acids are made to form a complex with copper. The ε-amino group of lysine which does not couple with copper is made to form ε-dinitropyridyl derivative of lysine with 2-chloro-3, 5-dinitropyridine. The excess pyridine is removed with ethyl acetate and the colour of ε-dinitropyridyl derivative is read at 390nm.

 

Materials and Reagents

 

Copper phosphate reagent

Pyridine reagent

0.05M Sodium Carbonate Buffer pH 9.0

Amino Acid Mixture

Sodium acetate buffer

Papain

1.2N HCl

Ethyl acetate

Centrifuge

Spectrophotometer

 

 

Composition of reaction mixtures

 

A.  Copper phospahte reagent

Solution A: Dissolve 2.89g of CuCI2.2H20 in 100ml water

Solution B: Dissolve 13.6g Na3P04.12H20 in 200 ml water

0.05M Sodium Borate Buffer pH 9.0

Pour solution A (100ml) to B (200ml) with swirling, centrifuge and discard the supernatant. Resuspend the pellet three times in 15ml sodium borate buffer and centrifuge after each suspension. After third washing resuspend the pellet, in 80ml of sodium borate buffer. The reagent must be prepared fresh for every seven days.

 

B.  Pyridine reagent

Dissolve 300mg 2-Chloro-3, 5-Dinitropyridine in 10 ml methanol. Prepare fresh just prior to use.

 

C.  0.05M Sodium Carbonate Buffer pH 9.0

 

D.  Amino Acid Mixture

Grind in a mortar 30mg alanine, 50mg glutamic acid, 60mg aspartic acid, 20mg cysteine, 300mg glutamic acid, 40mg glycine, 30mg histidine, 30mg isoleucine, 80mg leucine, 30mg methionine, 40mg phenylalanine, 80mg proline, 50mg serine, 30mg threonine, 30mg tyrosine and 40mg valine. Dissolve 100mg of this mixture in 10ml of sodium carbonate buffer (0.05M, pH 9.0)

 

E.  Papain solution

Dissolve 400mg technical grade papain (Sigma Co., USA) in 100 ml 0.1 M   sodium acetate buffer (pH 7.0)

 

F.  1.2N HCl

 

G.  Ethyl acetate

 

Procedure

 

1.  To 100mg of defatted grain sample, add 5ml of papain solution and incubate overnight at 65C. Cool to room temperature, centrifuge and decant the clear digest.

2.  To 1ml digest taken in a centrifuge tube, add 0.5ml carbonate buffer and 0.5ml copper phosphate suspension.

3.  Shake the mixture for 5 min in vortex mix and centrifuge.

4.  To 1ml supernatant, add 0.1ml pyridine reagent, mix well and shake for 2h.

5.  Add 5ml of1.2N HCI and mix.

6.  Extract three times with 5ml ethyl acetate and discard the ethyl acetate (top) layer.

7.  Read the absorbance of aqueous layer at 390nm.

8.  Prepare a blank with 5ml papain alone repeating steps 1 to 7.

9.  Dissolve 62.5mg lysine monohydrochloride in 50ml carbonate buffer (1mg lysine/ml). Pipette out 0.2, 0.4, 0.6, 0.8 and 1ml and make up to 1ml with sodium carbonate buffer. Add 4ml papain to each tube and mix. Pipette out one ml from each and add 0.5ml of aminoacid mixture and 0.5ml of copper phosphate suspension. Carry out steps 3 to 7. Prepare a standard curve from the readings of the standard lysine. The standard curve represents absorbance values for 40,80,120,160 and 200 μg lysine.

 

Calculation

 

Subtract the absorbance of the blank from that of the sample and calculate the lysine content in the aliquot from the graph.

 

                           Lysine value from graph in μg x 0.16

Lysine content of the sample =   -------------------------------------------- g per 16g N.

                      Percent N in the sample

 

 

 

Mertz E T, Jambunathan, R and Misra, P S (1975).In: Protein Quality, Agric Experiment stn Bull No 70 Purdue University USA p 11.