Sakaguchi Test: Objective, Principle, Procedure And Result Interpretation

What Is Sakaguchi Test?

The Sakaguchi test is a chemical test that includes a colorimetric reaction for the identification and quantification of the amino acid arginine. Arginine is an amino acid that has a guanidine group in its side chain, which is the C atom that binds N₂ with a single bond and binds N with a double bond. The test is named after the Japanese food Scientist and Organic Chemist, Shoyo Sakaguchi who described the test in 1925. The Sakaguchi reagent used in the test consists of 1-Naphthol and a drop of sodium hypobromite or sodium hypochlorite as an oxidizing agent. The guanidine group in arginine reacts with the Sakaguchi reagent to form a red-colored complex.

Objective

• To detect presence of amino acid guanidium group (R-NH-C=(NH2)+-NH2) i.e arginine in proteins.

Principle Of Sakaguchi Test

Sakaguchi test is based on the principle of reaction between 1-naphthol and the guanidinium groups in arginine, in the presence of an oxidizing agent. Sakaguchi test reaction is carried out using napththol and sodium hypobromite or sodium hypochlorite reagents. The guanidine group in arginine, which is oxidized by sodium hypochlorite will react with alpha-naphthol and produce red compounds under alkaline conditions. In this reaction, sodium hypobromite/hypochlorite acts as an oxidizing agent that facilitates the hydrogen bonding between two arginine molecules.

Experiment

Reagent And Material Required

Reagents

1. Test solutions
   1. Amino acids: 0.1% solution of amino acids like glycine, arginine, lysine, tyrosine etc.
   1. Protein solution: Egg albumin in distilled water (around 10%).
   1. Guanidines: 0.1% solutions of glycocyamine, methyl guanidine and creatine.
2. Sodium Hydroxide (NaOH): 40% (w/v).
3. Alpha-Naphthol: 1% in alcohol
4. Bromine water: Add a few drops of bromine to 100 ml water and shake. This should be done in a fume chamber or use 10% sodium hypochlorite solution.

Material Required

• Test tubes
• Test tube stand
• Pipettes

Sakaguchi Test Procedure

1. Mix 1ml of 40% NaOH with 3ml of the amino acid or test solution in a test tube.
2. Then, add two drops of 1-naphthol and mix thoroughly.
3. Add 4 to 5 drops of bromine water or add 1 to 2 drops of 10% sodium hypochlorite.
4. Observe the test tube for development of color.

Sakaguchi Test, Result Interpretation

• Positive Test: A positive Sakaguchi test result is indicated by the formation of red color. This confirms the presence of an arginine or guanidinium compound.
• Negative Test: A negative Sakaguchi result is indicated by absence of red color, and in this case, this confirms absence of arginine or a guanidinium compound.

Limitations Of Sakaguchi Test

• The test rate of Sakaguchi’s reaction is very slow and therefore, quantitative analysis of colored products is very much difficult with this test.
• Some of the guanidium groups in the solution might be destroyed by the hypochlorite, resulting in difficulties in testing results.