Sullivan and McCarthy’s Test- Definition, Principle, Procedure, Result, Uses

Methionine is one of the essential amino acids that humans cannot synthesize and must obtain from their diet. Methionine plays a vital role in various biological processes, such as protein synthesis, methylation, and sulfur metabolism. Methionine is also a precursor of other important molecules, such as cysteine, taurine, and S-adenosylmethionine. Therefore, the detection and quantification of methionine in biological samples is of great interest for biochemical and clinical studies.

Sullivan and McCarthy’s test is a simple and rapid method for the detection of methionine or methionine-containing proteins. The test is based on the formation of a red-colored complex between nitroprusside and methionine under alkaline conditions and subsequent acidification. The test has a high specificity for methionine as it does not react with other sulfur-containing amino acids like cysteine and cystine. The test can be performed with minimal equipment and reagents and can be applied to various types of samples, such as urine, blood, serum, tissue extracts, and fermented products.

In this article, we will discuss the definition, principle, procedure, result, uses, and limitations of Sullivan and McCarthy’s test. We will also provide some examples of how this test can be used in different contexts and applications. By the end of this article, you will have a clear understanding of what Sullivan and McCarthy’s test is and how it works.

Sullivan and McCarthy’s test is a biochemical test for the detection of the amino acid methionine or methionine-containing proteins. Methionine is one of the essential amino acids that cannot be synthesized by the human body and must be obtained from the diet. Methionine is also involved in various metabolic processes, such as protein synthesis, methylation, and sulfur metabolism. Therefore, it is important to detect the presence and amount of methionine in biological samples.

Sullivan and McCarthy’s test is a specific test for methionine which has a high degree of specificity towards methionine as it gives negative results with all other amino acids. The test, however, gives a positive result with glycylmethionine, which is a dipeptide composed of glycine and methionine. The test is based on the reaction between nitroprusside and alkaline solution of methionine under acidification. Nitroprusside is a complex compound that contains iron and cyanide ions. It reacts with methionine to form a red-colored complex that can be observed visually.

Sullivan and McCarthy’s test is a desirable method for methionine detection as it is a rapid and simple method for the assay of methionine even in fermented extract. The test can be performed in a few steps with minimal equipment and reagents. The test can also be modified for quantification where the test sample can be matched against the standard methionine solution. The test can be used for the detection of methionine either in a free form or in proteins. The test can also differentiate methionine from other sulfur-containing amino acids like cysteine and cystine.

The main objectives of Sullivan and McCarthy’s test are:

• To detect the presence of the amino acid methionine or methionine-containing proteins in a sample solution. Methionine is an essential amino acid that plays a role in protein synthesis, metabolism, and antioxidant defense. Methionine deficiency can cause various health problems such as liver damage, growth retardation, and neurological disorders. Therefore, it is important to determine the methionine content in food, feed, and biological samples.
• To differentiate methionine from other sulfur-containing amino acids like cysteine and cystine. Cysteine and cystine are also important amino acids that have various functions in the body such as maintaining the structure of proteins, forming disulfide bonds, and acting as antioxidants. However, they have different chemical properties and biological roles than methionine. Therefore, it is necessary to distinguish them from methionine in order to avoid confusion and errors in analysis.

The principle of Sullivan and McCarthy’s test is based on the reaction between nitroprusside and alkaline solution of methionine under acidification. Nitroprusside is a complex compound that contains five cyanide groups and one nitrosyl group. It has the ability to form colored complexes with various metal ions and organic compounds.

Methionine is an amino acid that contains a sulfur atom in its side chain. It can react with nitroprusside in an alkaline medium to form a purple complex. However, this complex is unstable and decomposes rapidly to form a red-colored compound. The structure of this compound is not known, but it is believed to be a thiocyanate derivative.

The reaction between methionine and nitroprusside can be represented by the following equation:

$$\text{CH}_3\text{SCH}_2\text{CH}_2\text{CH}(NH_2)\text{COOH} + \text{Na}_2 + 2\text{NaOH} \rightarrow \text{CH}_3\text{SCH}_2\text{CH}_2\text{CH}(NH_2)\text{COO}^- + \text{Na}^+ + ^- + \text{H}_2\text{O}$$

$$\text{CH}_3\text{SCH}_2\text{CH}_2\text{CH}(NH_2)\text{COO}^- + ^- \rightarrow \text{(Red compound)} + \text{(Other products)}$$

The red compound is soluble in water and can be detected by its characteristic color. However, other amino acids that contain sulfur, such as cysteine and cystine, can also react with nitroprusside and produce similar red compounds. Therefore, it is necessary to distinguish methionine from these amino acids.

One way to do this is to add acid to the reaction mixture. Acid hydrolysis destroys tryptophan, which also gives a red color with nitroprusside. Acid also converts cysteine and cystine into cysteic acid and cystamine, respectively, which do not react with nitroprusside. Thus, only methionine remains intact and gives a positive result.

Another way to differentiate methionine from other sulfur-containing amino acids is to add glycine to the reaction mixture. Glycine reacts with histidine, which also gives a red color with nitroprusside, and forms glycylhistidine. This compound does not react with nitroprusside and prevents the interference of histidine.

Therefore, by adding acid and glycine to the test solution, only methionine can give a positive result with nitroprusside. This makes Sullivan and McCarthy’s test a specific test for methionine detection.

The requirements for Sullivan and McCarthy’s test are as follows:

Reagents

• 10% sodium nitroprusside: This is the main reagent that reacts with methionine to produce a red color. It can be prepared by dissolving 10 g of sodium nitroprusside in 100 ml of distilled water.
• 5N sodium hydroxide: This is used to make the test solution alkaline. It can be prepared by dissolving 20 g of sodium hydroxide in 100 ml of distilled water.
• 6N hydrochloric acid: This is used to acidify the test solution after the addition of sodium nitroprusside. It can be prepared by diluting 50 ml of concentrated hydrochloric acid to 100 ml with distilled water.
• 2% glycine: This is used to eliminate the interference of histidine in the test. It can be prepared by dissolving 2 g of glycine in 100 ml of distilled water.

Sample solution

The sample solution can be any protein or amino acid solution that contains methionine or is suspected to contain methionine. The sample solution should be clear and free from any turbidity or precipitation. The sample solution can be prepared by dissolving or diluting the protein or amino acid in distilled water.

Materials required

• Water bath: This is used to heat the test solution at 40°C for 15 minutes.
• Ice bath: This is used to cool the test solution after heating.
• Vortex: This is used to mix the test solution and the reagents thoroughly.
• Test tubes: These are used to hold the test solution and the reagents. They should be clean and dry.
• Test tube stand: This is used to hold the test tubes upright and stable.
• Pipettes: These are used to transfer the test solution and the reagents accurately and precisely. They should be calibrated and rinsed before use.

The procedure of Sullivan and McCarthy’s test is as follows:

• Take 1 ml of the test solution in a test tube and add a few drops of 5N sodium hydroxide to make it alkaline.
• Add a few drops of 2% glycine solution and 10% sodium nitroprusside solution to the test tube and mix well by vortexing for 2 minutes.
• Place the test tube in a water bath at 40°C for 15 minutes to allow the reaction between methionine and nitroprusside to take place.
• Cool the test tube in an ice bath for 5 minutes and then add 0.5 ml of 6N hydrochloric acid to acidify the solution and destroy any tryptophan or histidine present.
• Mix the contents of the test tube by vortexing again and let it stand at room temperature for 15 minutes.
• Observe the color of the solution. A red color indicates a positive result for methionine, while no color change indicates a negative result.

The procedure is summarized in the following table:

The result of the Sullivan and McCarthy’s test depends on the color change of the solution after the addition of sodium nitroprusside and acid. The color change indicates the presence or absence of methionine in the sample.

• Positive result: The positive result is represented by the appearance of a red color. This indicates the presence of methionine or methionine-containing proteins in the sample. The red color is due to the formation of a complex between nitroprusside and methionine under acidic conditions.
• Negative result: The negative result is represented by the absence of a red color. This indicates the absence of methionine or methionine-containing proteins in the sample. The absence of color means that no complex was formed between nitroprusside and methionine.

The intensity of the red color may vary depending on the concentration of methionine in the sample. A higher concentration of methionine will produce a darker red color, while a lower concentration will produce a lighter red color. The test can be made quantitative by comparing the color intensity with a standard solution of known methionine concentration.

The result of the test should be interpreted with caution as some factors may interfere with the accuracy and reliability of the test. Some possible sources of error are:

• The presence of other amino acids that may react with nitroprusside and produce a false-positive result. For example, tryptophan and histidine can also produce a red color with nitroprusside under alkaline conditions. To avoid this, acid is added to hydrolyze any tryptophan present and glycine is added to eliminate any histidine present.
• The presence of impurities in the sample or reagents that may affect the color reaction. For example, commercial leucine may contain traces of methionine that may give a false-positive result. To avoid this, pure leucine should be used as a negative control.
• The variation in temperature, time, and pH that may affect the rate and extent of the color reaction. For example, higher temperature and longer time may enhance the color development, while lower temperature and shorter time may inhibit it. To avoid this, standard conditions should be followed as described in the procedure.

Sullivan and McCarthy’s test is a useful biochemical test for the detection and differentiation of methionine from other amino acids. Methionine is an essential amino acid that plays a vital role in various biological processes such as protein synthesis, methylation, and sulfur metabolism. Methionine is also a precursor of other important molecules such as cysteine, taurine, and S-adenosylmethionine. Therefore, the detection of methionine in biological samples can provide valuable information about the metabolic status and health condition of an organism.

Some of the uses of Sullivan and McCarthy’s test are:

• The test can be used to detect the presence of methionine or methionine-containing proteins in various samples such as blood, urine, cerebrospinal fluid, milk, cheese, meat, eggs, etc. The test can also be used to identify methionine-producing bacteria or fungi in culture media or environmental samples.
• The test can be modified for quantification where the test sample can be matched against a standard methionine solution. The intensity of the red color can be measured by a spectrophotometer or a colorimeter and compared with a standard curve to determine the concentration of methionine in the sample. This can help to assess the level of methionine intake or excretion in humans or animals.
• The test can be used to study the effect of various factors such as diet, drugs, diseases, stress, etc. on the methionine metabolism and homeostasis in different organisms. For example, the test can be used to monitor the response of patients with homocystinuria or cystathioninuria to dietary or pharmacological interventions that aim to lower the plasma levels of homocysteine or cystathionine, respectively. These are two toxic metabolites that accumulate due to defects in the methionine cycle.
• The test can be used to investigate the role of methionine in various physiological and pathological processes such as aging, oxidative stress, inflammation, cancer, neurodegeneration, etc. For example, the test can be used to measure the changes in methionine levels in response to oxidative stress induced by hydrogen peroxide or ultraviolet radiation. The test can also be used to evaluate the effect of methionine restriction or supplementation on the lifespan and healthspan of different organisms.

• The test might give a false positive result with commercial leucine as it might contain some traces of methionine. This can be avoided by using pure leucine or by performing a confirmatory test for methionine such as the Sakaguchi test.
• The test might not give reproducible results with standard solutions of methionine. This can be due to the variation in the concentration of the reagents, the temperature and time of incubation, and the quality of the nitroprusside. To ensure accuracy and consistency, the test should be performed under standardized conditions and with fresh reagents.
• The test is not specific for methionine as it also gives a positive result with glycylmethionine. This can be a problem if the sample contains both methionine and glycylmethionine. To overcome this limitation, the sample can be treated with an enzyme that hydrolyzes glycylmethionine such as carboxypeptidase A before performing the test.
• The test is not sensitive enough to detect low levels of methionine. The red color might not be visible if the sample contains less than 0.1% of methionine. To increase the sensitivity of the test, the sample can be concentrated by evaporation or precipitation before performing the test.

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