Nitroprusside’s Test: Principle, Reaction, Reagents, Procedure and Result Interpretation

Nitroprusside’s Test

(Detection of Cysteine)

The nitroprusside’s test is specific for cysteine, the only amino acid containing a sulfhydryl group (—SH). The group reacts with nitroprusside in an alkaline solution to yield a red complex. Methionine does not react to this test because it has got thioester linkage which is difficult to break. Nitroprusside test is commonly used to detect the ketones in the urine.

Principle:

This test is specific for Cys which has a free sulfhydryl group (-SH) that is able to react with the nitroprusside in the presence of excess ammonia (NH4OH). Principally, the free thiol group of Cys gives a red color with sodium nitroprusside in the presence of NH₄OH.

Reactions:

Fig: Na-nitroprusside reaction with Cys amino acid.

Reagents:

• Sulphur amino acids (1.0% cystine, and methionine)
• Sodium nitroprusside (2% prepared fresh)
• Ammonium hydroxide

Procedure:

• Put 2 mL amino acid solution into the test tube.
• Add 0.5 mL nitroprusside solution and shake thoroughly.
• Add 0.5 mL ammonium hydroxide.
• Observe the color change.

Result Interpretation:

Positive test: Formation of red color indicate presence of cysteine.

Negative test: No formation of red color indicate absence of cysteine.

References:

• http://www.biologydiscussion.com/proteins/qualitative-and-quantitative-tests-for-amino-acids-and-proteins/13065
• http://ecoursesonline.iasri.res.in/mod/page/view.php?id=4188
• http://www.chem.boun.edu.tr/wp-content/uploads/2014/04/Chem-415-Experiment-2.pdf