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![Specific rotation
•specific rotation – the angle of rotation of plane
polarized light by a 1.00 gram per cm-3 sample in a
1 dm tube. [α ]D (D = sodium lamp, λ = 589 mμ).
α
•[ α ]D = ------------- where α = observed
rotation
l * d
l = length (dm)
d = concentration (g/cc)](https://image.slidesharecdn.com/aminoacids-180710135906/75/Amino-acids-ppt-Sumati-s-Biochemistry-9-2048.jpg)
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Amino acids ppt |Sumati's Biochemistry|
- 1. • Proteins arepolymers of simple units known as amino acids.
- 2. AMINO ACIDS ASPRECURSORS Apart from being building blocks of proteins many amino acids are used to synthesize other molecules, for example: • Serotonin from Tryptophan . • Catecholamine dopamine, epinephrine and norepinephrine from Tyrosine (and its precursor phenylalanine). • Porphyrins such as heme from Glycine . • Nitric oxide from Arginine . • Polyamines from Ornithine and S-adenosylmethionine. • Nucleotides from Aspartate, glycine, and glutamine. • Various phenylpropanoids from Phenylalanine.
- 3. GENERAL ANATOMY OFAMINO ACIDS • General anatomy of an amino acid. Molecule of an amino acid containscarboxyl(COOH) and amino (NH2) functional groups, and each molecule features a different side chain, also known as R group, which vary greatly in properties.
- 4. Stereoisomerism •The subject ofIsomerism is divided into two categories • structural isomerism •stereoisomerism . •Stereoisomerism is exhibited in two forms- •(a) Geometrical isomerism •(b) Optical Isomerism.
- 5.
- 6. •(b)Optical isomers- •Optical isomerismis commonly found when a molecule contains one or more chiral (Greek cheir= hand) or asymmetric carbon atoms.
- 7.
- 8. PROPERTY OF OPTICALROTATION
- 9. Specific rotation •specific rotation– the angle of rotation of plane polarized light by a 1.00 gram per cm-3 sample in a 1 dm tube. [α ]D (D = sodium lamp, λ = 589 mμ). α •[ α ]D = ------------- where α = observed rotation l * d l = length (dm) d = concentration (g/cc)
- 10. Stereoisomerism in aminoacids and comparison with glyceraldehyde (A)D- Glyceraldehyde (B)D-Serine (C)L- Glyceraldehyde (D)L- Serine (E)L- Alanine (F)D- Alanine
- 11. Classifications • Amino acidsare classified according to lots of different features as follows. • (1) One of them is whether or not human beings can synthesize them in the body. Actually all twenty of them are necessary for human health. • 3 types are: • Nonessential, Essential Semiessential • Asparagine, Leucine, Histidine • Alanine, Isoleucine • Arginine, Lysine • Aspartic acid, Threonine • Cysteine Methionine • Glutamic acid, , Phenylalanine • Glutamine, Valine • Proline Tryptophan • Glycine • Tyrosine, • Serine
- 12. Classification (2)One more classificationdepends on the side chain structure is given as follows: • containing sulfur (Cysteine and Methionine) • neutral (Asparagine, Serine, Threonine, and Glutamine) • acidic (Glutamic acid and Aspartic acid) and basic (Arginine and Lysine) • aliphatic (these include Leucine, Isoleucine, Glycine, Valine, and Alanine • aromatic (these include Phenylalanine, Tryptophan, and Tyrosine)
- 13. Classification (3)Classification based onpolarity of R group
- 14. Classification (A) Amino acidswith nonpolar or hydrophobic R groups Side chain(R) groups Name of amino acid Three alphabet code One alphabet code Non polar Alanine Ala A Valine Val V Leucine Leu L Isoleucine Ile I Methionine Met M Phenylalanine Phe F Tryptophan Trp W Proline Pro p
- 15. (B)Amino acids withpolar but uncharged R groups Side chain(R) groups Name of amino acid Three alphabet code One alphabet code Aliphatic (a)containing hydroxyl group Serine Ser S Threonine Thr T (b)Containing sulfhydryl group Cysteine Cys C (c)Containing amide groups Aspargine Asn N Glutamine Gln Q Aromatic (a)Containing hydroxyl group Tyrosine Tyr Y Without side chain(R) group Glycine Gly G
- 16. (C) Amino acidswith positively charged groups Side chain(R) groups Name of amino acid Three alphabet code One alphabet code Positively charged (a) Containing second amino group Lysine Lys L (b)Containing guanidino group Arginine Arg R (c) Containing imidazole group Histidine His H
- 17. (D)Amino acids withnegatively charged R groups Side chain(R) groups Name of amino acid Three alphabet code One alphabet code Negatively charged (a) Containing second carboxylic group Aspartic acid Asp D Glutamic acid Glu E
- 18.
- 19. Amino acids aszwitterions AMINO ACID
- 20. Titration of anamino acid
- 23. Chemical reactivity ofthe amino acids •Functional groups in amino acids can take part in many chemical reactions: • Carboxyl group– can form esters, amides and amines, and undergo decarboxylation • Amino group– can undergo acylation, amidation and esterification • Hydroxyl and sulfhydryl groups– can undergo oxidation and esterification
- 24. Reactions involving COOHgroup •Reaction with alkalies (Salt formation) •Reaction with alcohols (Esterification ). •Reaction with amines
- 25. Reactions involving NH2group •Reaction with mineral acids (Salt formation) •Reaction with formaldehyde •Reaction with benzaldehyde •Reaction with nitrous acid(Van Slyke reaction) •Reaction with acylating agents (Acylation)- •Reaction with FDNB or Sanger’s reagent
- 26. Reactions involving bothCOOH and NH2 group •Reaction with triketohydrindene hydrate (Ninhydrin reaction)- •Reaction with phenyl isocyanate-. •Reaction with phenyl isothiocyanate or Edman reagent. •Reaction with phosgene- •Reaction with carbon disulfide
- 27. Reactions involving Rgroup or side chain • Biuret Test: • formation of coordination complex •The Biuret Test- works for any compound containing two or more of the following groups.
- 28. • Xanthoproteic Test:Phenyl rings containing an activating group can be nitrated producing a yellow product. • Pauly's diazo Test: This test is specific for the detection of Tryptophan or Histidine. • Millon's test: Phenolic amino acids such as Tyrosine and its derivatives respond to this test. • Histidine test : This test was discovered by Knoop. This reaction is specific for histidine. • Hopkins Cole test: This test is specific test for detecting tryptophan. • Sakaguchi test:This test is specific for detecting Arginine. • Lead sulphide test : This test is specific for Sulphur containing amino acids, such as cysteine and cystine . • Nitroprusside test: This test is specific for cysteine and methionine . • Isatin test:This test is specific for Imino acids such as Proline and hydroxyproline .
- 29. References • http://www.chemexplore.net/symmetry.htm • http://www.chemguide.co.uk/basicorg/isomerism/optical.html •http://curvedarrows.blogspot.in/ • BCH4053L Biochemistry Laboratory Manual • www.molecularstation.com • http://people.uwplatt.edu/~sundin/351/351h-pro.htm
Editor's Notes
- #2 Proteins are biomolecules and fundamental constituents of every cell in the living world. These have nitrogen as an essential component and are synthesized under strict control of the genes. They show a large diversity of size, shape and biological function ,and perform various functions. Out of hundreds of amino acids found in nature only twenty amino acids actually synthesize proteins in our body . Amino are associated in various combinations and numbers to make different proteins .Thus properties and functions of a protein depend on its amino acid composition. Amino acids act as: enzymes (catalysts), metabolic intermediates, carriers of energy and waste products and hormones
- #3 AMINO ACIDS AS PRECURSORS Apart from being building blocks of prteins many amino acids are used to synthesize other molecules, for example: Tryptophan is a precursor of the neurotransmitter serotonin. Tyrosine (and its precursor phenylalanine) are precursors of the catecholamine ,neurotransmitters dopamine,epinephrine and norepinephrine. Glycine is a precursor of porphyrins such as heme. Arginine is a precursor of nitric oxide. Ornithine and S-adenosylmethionine are precursors of polyamines. Aspartate, glycine, and glutamine are precursors of nucleotides. Phenylalanine is a precursor of various phenylpropanoids, which are important in plant metabolism.
- #4 General anatomy of an amino acid. Molecule of an amino acid contains carboxyl (COOH) and amino (NH2) functional groups, and each molecule features a different side chain, also known as R group, which vary greatly in properties.. Except for proline and its derivatives, all of the amino acids commonly found in proteins possess this type of structure. All 20 amino acids in pure form are white, crystalline, high-melting.( - up to 200-300°C) solids, able to dissolve in water . They only dissolve sparingly in organic solvents, and the extent of their solubility depends on the size and nature of the side chain, and other properties vary for each particular amino acid.
- #5 The study of amino acids immediately introduces the subject of stereoisomerism which is a kind of Isomerism found among organic molecules . The subject of Isomerism is divided into structural and stereoisomerism . In structural isomerism isomers have same molecular formula but they differ from each other by having different structures. Stereoisomers share common molecular and structural formula but they differ from each other in configuration .Configuration of a molecule is arrangement of atoms in space. Stereoisomerism is exhibited in two forms- (a) Geometrical isomerism (b) Optical Isomerism.
- #6 Fumeric acid is trans isomer of cis isomer Maleic acid .
- #7 Optical isomerism is commonly found when a molecule contains one or more chiral (Greek cheir= hand) or asymmetric carbon atoms.Van’t Hoff LeBel introduced the concept of tetrahedral carbon atom. In many compounds carbon nucleus sits in the center of the tetrahedron and the four covalent bonds or bond axes extend out to the corners of the tetrahedron.When four different groups are attached to those bonds,the carbon atom in the center of the molecule is known as chiral center ( or a chiral carbon tom). n chiral centers 2n maximum stereoisomers .
- #8 These groups may be arranged in space in two different ways so that two different compounds are formed which can not be superimposed on each other.Instead, one compound is related to the other as right hand is related to left hand . These compounds are said to possess “handedness” and are mirror images of each other.Mirror image isomers constitute an enantiomeric pair, one member of the pair is enantiomer of the other
- #9 Enantiomers have the same physical properties and cannot be separated by normal separation techniques like distillation, etc, but differ in reaction with optically active reagents.Enantiomers can rotate the plane of polarised light to clockwise or anticlockwise. A solution of one enantiomer rotates the plane of polarisation in a clockwise direction. This enantiomer is known as the{ (+)form; d } or dextrorotatory. A solution of the other enantiomer rotates the plane of polarisation in an anti-clockwise direction. This enantiomer is known as the {(-) form; l} or levorotatory .For example, one of the optical isomers (enantiomers) of the amino acid alanine is known as (+)alanine.The other enantiomer of alanine is known as or (-)alanine.If the solutions are equally concentrated the amount of rotation caused by the two isomers is exactly the same but in opposite directions.When equally concentrated solutions of (+)and (-) forms are mixed together they are known as a racemic mixture or racemate. It has no effect on plane of polarised light.
- #10 The compounds showing property of optical rotation are known as optically active compounds .The angle of rotation of plane polarized light by an optically active substance is proportional to the number of atoms in the path of the light.Optical activity is measured by the instrument polarimeter,and it is expressed in term of specific rotation as follows- specific rotation – the angle of rotation of plane polarized light by a 1.00 gram per cm-3 sample in a 1 dm tube. [α ]D (D = sodium lamp, λ = 589 mμ). α [ α ]D = ------------- where α = observed rotation l * d l = length (dm) d = concentration (g/cc)
- #11 Except for glycine (optically inactive) all amino acids have at least one chiral center and show optical isomerism. The L-/D- isomers are specified with reference to the configurations of D-glyceraldehyde and the closely related amino acid, serine. On Comparing the orientations of -OH on C-2 of D-glyceraldehyde with that of NH3+on the α–C atom i.e.C-2 of Ser, the two functional groups are in opposite directions so the amino acid is designated as L-Ser.All other amino acids are compared to Ser and the subscript‘s’ is sometimes used as in L-Alanine.In order to write the formula in the correct stereospecific configuration, the –COO group is to be written above the α-C atom when -NH3+is written to the left. The L-amino acids are prevalent in proteins
- #12 Amino acids are classified according to lots of different features as follows. (1) One of them is whether or not human beings can synthesise them in the body .Thus they may be of 3 types: Nonessential, Essential, Conditionally essential Actually all twenty of them are necessary for human health. Those 8 called essential (or indispensable) can't be produced by the body and therefore should be supplied by food: Leucine, Isoleucine, Lysine, Threonine, Methionine, Phenylalanine, Valine, and Tryptophan. One more amino acid, Histidine, can be considered semi-essential, as the human body doesn't always need dietary sources of it. Meanwhile, conditionally essential amino acids aren't usually required in the human diet, but are able to become essential under some circumstances. Finally, nonessential ones are produced by the human body either out of the essential ones or from normal proteins breakdown. These include Asparagine, Alanine, Arginine, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Proline, Glycine, Tyrosine, and Serine.
- #13 (2)One more classification depends on the side chain structure is given as follows: containing sulfur (Cysteine and Methionine) neutral (Asparagine, Serine, Threonine, and Glutamine) acidic (Glutamic acid and Aspartic acid) and basic (Arginine and Lysine) alphatic (these include Leucine, Isoleucine, Glycine, Valine, and Alanine) aromatic (these include Phenylalanine, Tryptophan, and Tyrosine)
- #14 (3) classification based on the polarity of R group is more meaningful as it depicts the possible functional roles of amino acids in proteins.Thus amino acids are (A)nonpolar or hydrophobic (B) polar but uncharged (C)polar because of negative charge at physiological pH; 7.4 (D)polar because of positive charge at physiological pH. Characterstic features of amino acids belonging to above given classification are written below along with three letter and one letter nomenclature code-
- #15 (A)Amino acids with nonpolar or hydrobhobic R groups are categorised as follows- * Amino acids with aliphatic R groups in this category are Alanine (Ala ; A) ,Valine (Val ;V) ,Leucine(Leu ; L) ,Isoleucine (Ile ;I) , and Methionine (Met ; M) * Amino acids with aromatic R groups in this category are Phenylalanine (Phe ; F),and Tryptophan (Trp ;W) *Unusual amino acid Proline(Pro ; P) also belongs to this group and its nitrogen atom is present in the structure as a secondary amine rather than as a primary amine.
- #16 (B)Amino acids with polar but uncharged R groups are categorised as follows- *Amino acids with aliphatic R groups in this category - can take part in hydrogen bonding.Several amino acids contain hydroxyl group (Serine ;Ser; S , Threonine ;Thr ; T ,) or sulfhydryl group (Cysteine ;Cys ;C) and other two have amide groups (Aspargine ;Asn ;N ,Glutamine ;Gln ; Q). *Amino acid with aromatic R group in this category is Tyrosine( Tyr ; Y) which contains hydroxyl group available for hydrogen bonding. *Glycine (Gly ;G) which does not have any R group is kept in this category of amino acids. It shows polar behaviour due to the presence of polar amino and carboxyl groups contributing the large part of the mass of the molecule.
- #17 (C) Amino acids with positively charged groups- This group includes following three amino acids---- *Amino acid Lysine(Lys ;L) contains second amino group(epsilon ;ɛ) having pka=10.5.It remains in more than 50% positively charged state at any pH below the pka of that group. *Amino acid Arginine (Arg ;R) contains strongly basic guanidino group(pka=12.5). *Amino acid Histidine (His ;H) is weakly basic because of the presence of imidazole group (pka=6.0).Histidine is the only amino acid which contains a proton which dissociates in the neutral pH range.This feature allows histidine to play important role in catalytic activities of certain enzymes.
- #18 (4)Amino acids with negatively charged R groups This group includes following two dicarboxylic amino acids- *Aspartic acid(Asp ;D) having pka=3.9 of second carboxylic group at neutral pH. *Glutamic acid(Glu ;E) having pka=4.3 of second carboxylic group at neutral pH Second carboxyl group of both amino acids dissociates at neutral pH to give net -1 charge to the molecules.
- #19 A carbon atom can form up to 4 covalent bonds. If one carbon bonds to more than 2 other carbons, a branch forms. Valine, Leucine, and Isoleucine are called branched-chain amino acids - their side-chains have a branch. ( the combination of these 3 amino acids represent around one-third of skeletal muscle in our body).
- #20 An amino acid has both a basic amine group and an acidic carboxylic acid group. At the physiological pH (7.4) amino acids exist as zwitterions – positive and negative charge on the same molecule. There is an internal transfer of a hydrogen ion from the -COOH group to the -NH2 group to leave an ion with both a negative charge and a positive charge. This is called a zwitterion. This is the form that amino acids exist in even in the solid state. If you dissolve the amino acid in water, a simple solution also contains this ion. A zwitterion is a compound with no overall electrical charge, but which contains separate parts which are positively and negatively charged. Isoelectric point (pI or IpH) of the amino acid is the pH at which the net charge on the amino acid is zero. At this point the amino acid is a zwitterion and is electrically neutral.
- #21 When a weak acid, HA, is titrated with an alkali, then at the point of half neutralization of HA, the titration curve shows an inflection and pH of the solution equals pKa for the acid. Since an amino acid has at least two acidic groups at extremely low pH, its titration curve shows two inflection points with separate pK values for –COOH and -NH3+ . Examine the graph (titration of diprotic glycine with NaOH) from left to right. The 1st inflection point (pK1 = 2.3) is due to dissociation of the stronger acidic group, –COOH, while the 2nd inflection point (pK2 = 9.6) is due to NH3+ . At pH 6.0 the graph shows a change in direction. This is the pH at which -COOH has been completely dissociated to –COO- while NH3+ still remains undissociated. It is the isoelectric point (pI or IpH) of the amino acid i.e. the pH at which the net charge on the amino acid is zero. . It is possible to predict the ionic species present at pH 1, 2,3 ,4, 5,6,7,8, 9.6 and 12 in the titration curve with alteration in the charge of an amino acid in a changing pH mileau.
- #22 These are the titration curves of glutamic acid and lysine and we can see 3 pka values for both the acids. pka1 is for dissociation of carboxylic acid pka2 is for dissociation of amino group. pka3 is for ionisable group in the R group.
- #23 This the titration curve of His, in which the ‘R’ group is charged; have three pK values. pK 1is for the carboxylic group, pK2 is for the amino group and pKR is for an ionizable group in the ‘R’ chain. Each amino acid has its own specific pI and this varies with the nature of the medium. The behaviour of a peptide/protein in the pH of body fluids or in extraction media, depends on the pK values of its constituent amino acids. In summary, titration curves are helpful in the identification of amino acids as follows: The number of pKa values differentiates polar and nonpolar amino acids from charged amino acids. The position of the pKa values for charged amino acids allows one to identify positively charged from negatively charged amino acids. Comparisons between experimental and literature pKa values can allow the identification of a specific amino acid.
- #24 Chemical reactivity of the amino acids Functional groups in amino acids can take part in many chemical reactions Carboxyl group– can form esters, amides and amines, and undergo decarboxylation • Amino group– can undergo acylation, amidation and esterification • Hydroxyl and sulfhydryl groups– can undergo oxidation and esterification Specific reactions of the amino and carboxylic groups are important in the body and in protein analysis. Decarboxylation of the amino acids in digestion and metabolism gives amines. Oxidative deamination in the liver and kidney produces the corresponding keto acids. Free amino groups in the globin part of hemoglobin attach CO2 to give carbamino derivatives that transport CO2 in the blood
- #25 Reactions involving COOH group Reactions with alkalies (Salt formation) – The carboxylic group of amino acids can release a H+ ion with the formation of carboxylate (COOH) ions. These may be neutralised by cations like Na+ and Ca++ to form salts. Thus amino acids react with alkali to form salts. Sodium salt of glutamic acid (monosodium glutamate) is used commercially as a flavouring –enhancing agent. .Reaction with alcohols (Esterification )-With alcohols,corresponding esters are produced. Reaction with amines-Amino acids react with amines to form amides.
- #26 Reactions involving NH2 group 1-Reaction with mineral acids (Salt formation)-When either free amino acid or proteins are treated with mineral acids like HCl, the acid salts are formed.The basic amino acids, arginine and lysine react with CO2 in the presence of air to form carbonate salts ,and are stored in the form of their monochlorides. 2-Reaction with formaldehyde-With formaldehyde,the hydroxy-methyl derivatives are formed.These derivatives are insoluble in water and resistant to attack by micro-organisms. The reaction is the basis of the Sorenson formol titration method for determining the purity of the individual amino acids. 3-Reaction with benzaldehyde-Schiff’s bases are formed in this reaction 4-Reaction with nitrous acid(Van Slyke reaction)-The amino acids react with HNO2 to liberate N2 gas and to produce the corresponding α-hydroxy acids. 5-Reaction with acylating agents (Acylation)-Amino acids in alkaline medium react with many acid chlorides(CH3.COCI,C6H5.COCI)and acid anhydrides(CH3.CO-O-OC.CH3,phthalic anhydride). 6-Reaction with FDNB or Sanger’s reagentFDNB(1-fluoro-2,4-dinitrobenzene)reacts with amino acids to procduce yellow coloured derivatives,DNB-amino acids.
- #27 Reactions involving both COOH and NH2 group Reaction with triketohydrindene hydrate (Ninhydrin reaction)-Ninhydrin(=indane-1,2,3,-trione hydrate)is a powerful oxidising agent and causes oxidative decarboxylation of α-amino acids producing CO2,NH3 and an aldehyde with one less carbon atom than the parent amino acid.The reduced ninhydrin (or hydrindantin) then reacts with liberated NH3and a molecule of ninhydrin forming blue coloured complex.Van Slyke used this equation for quantitative measurement of amino acids. CO2 released is measured manometrically(indicative of presence of amino acids only).The reaction is sensitive and gives reliable results.Proline and hydroxyl proline (imino acids)give yellow colour in this reaction while other amines(not α- amino acids)react to give blue colour too but without evolving CO2 . Reaction with phenyl isocyanate-With phenyl isocyanate, hydantoic acid is formed which in turn can be converted to hydrantoin. Reaction with phenyl isothiocyanate or Edman reagent- Phenyl isothiocyanate reacts with amino acids to give thiohydantoic acid. On treatment with acids in non hydroxylic solvents, the latter cycliize to thiohydantoin. Reaction with phosgene-With phosgene amino acids form N-carboxyanhydride. Reaction with carbon disulfide-With carbon disulfide amino acids form 2-thio-5-thiozolidone.
- #28 Reactions involving R group or side chain Biuret Test: The Biuret Test is a general test for proteins. When a protein reacts with copper(II) sulfate (blue)(Biuret reagent; alkaline 0.2% copper sulphate solution), the positive test is the formation of a violet colored co-ordination complex.The colour deepens as the number of peptide bonds is increased.The Biuret Test works for any compound containing two or more of the following groups.
- #29 Xanthproteic Test: Phenyl rings containing an activating group can be nitrated producing a yellow product. The production of a yellow colored product upon the addition of nitric acid is a test for the presence of tyrosine or tryptophan in a protein. The addition of strong base will deepen the color to orange due to the ionization of the phenolic nitro-derivatives. The yellow stains on the skin caused by nitric acid are the result of the xanthoproteic reaction. Pauly's diazo Test: This test is specific for the detection of Tryptophan or Histidine. The reagent used for this test contains sulphanilic acid dissolved in hydrochloric acid. Sulphanilic acid upon diazotization in the presence of sodium nitrite and hydrochloric acid results in the formation a diazonium salt. The diazonium salt formed couples with either tyrosine or histidine in alkaline medium to give a red coloured chromogen (azo dye). Millon's test Phenolic amino acids such as Tyrosine and its derivatives respond to this test. Compounds with a hydroxybenzene radical react with Millon’s reagent to form a red colored complex. Millon’s reagent is a solution of mercuric sulphate in sulphuric acid. Histidine test This test was discovered by Knoop. This reaction involves bromination of histidine in acid solution, followed by neutralization of the acid with excess of ammonia. Heating of alkaline solution develops a blue or violet coloration. Hopkins cole test This test is specific test for detecting tryptophan. The indole moiety of tryptophan reacts with glyoxilic acid in the presence of concentrated sulphuric acid to give a purple colored product. Glyoxilic acid is prepared from glacial acetic acid by being exposed to sunlight. Sakaguchi test Under alkaline condition, α- naphthol (1-hydroxy naphthalene) reacts with a mono-substituted guanidine compound like arginine, which upon treatment with hypobromite or hypochlorite, produces a characteristic red color. Lead sulphide test Sulphur containing amino acids, such as cysteine and cystine. upon boiling with sodium hydroxide (hot alkali), yield sodium sulphide. This reaction is due to partial conversion of the organic sulphur to inorganic sulphide, which can detected by precipitating it to lead sulphide, using lead acetate solution. Nitroprusside test Red colour develops with sodium nitroprusside in dilute ammonium hydroxide. This test is specific for cysteine. Addition of sodium nitroprusside[Na2Fe(CN)5NO] to an alkaline solution of methionine followed by the acidification of the reaction yields a red colour. This reaction also forms the basis for the quantitative determination of methionine. Isatin test Imino acids such as Proline and hydroxyproline condense with isatin reagent under alkaline condition to yield blue colored adduct