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A MODIFIED PHOTOMETRIC NINHYDRIN METHOD THE ANALYSIS OF AMINO AND IMINO ACIDS BY FOR WALTER TROLL* New AND R. KEITH CANNAN College of Medicine, New (From the Department of Chemistry, York, New York University York) (Received for publication, June 18, 1952) Primary amino acids may be quantitatively deaminated by ninhydrin with the formation of carbon dioxide and an aldehyde. Under some conditions, the ammonia appears as such, but, under others, it condensesto a greater or lesser exte
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  A MODIFIED PHOTOMETRIC NINHYDRIN METHOD FORTHE ANALYSIS OF AMINO AND IMINO ACIDS BY WALTER TROLL* AND R. KEITH CANNAN(From the Department of Chemistry, New York University College of Medicine, NewYork, New York) (Received for publication, June 18, 1952) Primary amino acids may be quantitatively deaminated by ninhydrinwith the formation of carbon dioxide and an aldehyde.Under some con-ditions, the ammonia appears as such, but, under others, it condenses o agreater or lesser extent with the reagent to form diketohydrindylidene-diketohydrindamine (DYDA).The familiar violet color which is associ-ated with the reaction of amino acids with ninhydrin is attributed to theanion of this compound.Ruhemann (l-3) was of the opinion that thefirst step in the reaction is the oxidative deamination of the amino acidwith the formation of a molecule of ammonia and a molecule of the reduc-tant of ninhydrin, diketohydrindol, in the form of the meriquinone, hydrin-dantin. The ammonia then condenses with the hydrindantin to formDYDA. This scheme is consistent with the fact that ammonia formsDYDA with ninhydrin only in the presence of a reducing agent capableof producing some hydrindantin. It is inconsistent, however, with thefact that ammonia reacts more slowly and less completely than do aminoacids under the sameconditions. One must conclude that the deaminationand the condensation are coupled in some fashion.Moore and Stein (4) have recently developed the reaction into a rapidand convenient photometric method for the estimation of amino acids.They showed that the low and variabIe yields of color usually obtainedwere due to the autoxidation of some of the diketohydrindol formed as aresult of the oxidative deamination. Introduction of a reducing agent intothe system materially increased the amount of color and made it possibleto obtain consistent and reproducible results, Even so, in the procedureof Moore and Stein, the yield of color per molecule varies with the aminoacid, and, in no case, s as great as that of a molecule of DYDA.Seeking an explanation for the apparent lack of stoichiometry of thereaction, we were impressed with the observation of Moore and Stein thatthe colors formed in their procedure slowly faded on standing at roomtemperature over a period of hours. Since the colors are developed byexposure of the reaction system to 100” for 20 minutes, it was reasonable * Present address, May Institute for Medical Research of the Jewish HospitalAssociation, Cincinnati, Ohio.803  b  y  g u e s  t   , onM ar  c h  8  ,2  0 1 2 www. j   b  c . or  gD  ownl   o a d  e d f  r  om   804 NINHYDRIN METHOD to infer that some destruction of DYDA occurred in this step. We, there-fore, sought conditions under which the rate of formation of DYDA wasincreased relative to its rate of destruction. Organic solvents such asalcohol, dioxane, methyl Cellosolve, pyridine, and phenol were found toaccelerate the development of color to varying degrees. Ultimately aphenol-pyridine system was adopted as the most effective solvent. In thissystem containing 5 per cent water the majority of the amino acids gavequantitative yields of color in 20 minutes at room temperature (Table I). TABLE IColor Recoveries Obtained at Room Temperature0.01 ml. of amino acid, 0.002 M, 2 ml. of 80 per cent phenol in alcohol, 0.5 ml. ofpyridine, 500 mg. of ninhydrin, and 100 mg. of hydrindantin are allowed to react for20 minutes at room temperature, and diluted to 10 ml. with 60 per cent alcohol and1 per cent formaldehyde. Amino acid Glycine. ...................................Alanine ....................................Valine .....................................Leucine ...................................Isoleucine .................................Phenylalanine .............................Tyrosine ..................................Tryptophan ...............................Serine .....................................Threonine .................................Methionine ................................Glutamic acid .............................Aspartic “ .............................Histidine ..................................Arginine ...................................-1 P’ X extinction (570 mp er cent yield of DYDA 10.00 46.521.2 98.421.5 99.521.7100.521.4 99.010.3 48.012.2 55.514.265.720.5 95.021.2 98.421.5 99.521.7 100.59.25 42.714.6 68.021.8 101 .o Exposure to 100” for 3 to 5 minutes gave quantitative yields of color for allthe amino acids except tryptophan and lysine (Table II) in a system con-taining 20 per cent water.The imino acids react with ninhydrin in an entirely different fashion.Carbon dioxide is evolved and yellow colors are formed very rapidly, evenat room temperature and at pH 7. Reducing agents are without influence.Deamination does not occur and the imino acid residue condensesdirectlywith the ninhydrin to form the pigment.Grassmann and von Arnim (5) have isolated red derivatives of prolineand hydroxyproline and have identified them as di(diketohydrindylidene)-pyrroles. We have prepared these compounds and have observed that in  b  y  g u e s  t   , onM ar  c h  8  ,2  0 1 2 www. j   b  c . or  gD  ownl   o a d  e d f  r  om   1%‘. TROLL AND R. K. CANNAN 805aqueous solution they readily change to yellow products possessing a broadabsorption with a maximum at 440 rnp. They are not well suited tophotometry because of the relatively large absorption of ninhydrin in thisregion of the spectrum, The red compounds, on the other hand, exhibitintense absorption in the region 550 to 570 rn~. TABLE II Results with Recommended Method for Amino Acids Amino acid Glyeine ................................... Alanine ....................................Valine ..................................... Leucine .................................. Isoleucine .................................Phenylalanine ............................. Tyrosine .................................. Tryptophan ...............................Serine .....................................Threonine .................................Methionine ................................Glutamic acid .............................Aspartic “ .............................Histidine ..................................Arginine ...................................Lysine ..................................... Sarcosine ..................................Ammonia ..................................Leucylglycine. ............................. Glycylglycylleucine. ....................... Alanylglycylglycylleucine .................. Urea, creatinine, aniline, p-aminobenzoicacid, hippuric acid ....................... -/I ud extinction (570 mp) -.- I I ‘er cent yield of DYDA 21.122.021.621.821.621.821.316.321.422.122.021.421.322.021.123.85.26.320.619.318.30.0__-98102.0100100.1100100.198.875.499.0102.6102.099.098.6102.098.0110.524.629.295.589.585.0 It is the yellow products of the imino acids that are formed when theprocedure of Moore and Stein is applied to a protein hydrolysate. Wehave demonstrated, however, that the red compounds are fugitive inter-mediates. It has been possible to obtain quantitative recovery of the redderivative of hydroxyproline by continuous extraction of an appropriatereaction system with benzene. Under the conditions to be described, thered pigment of proline forms more slowly and is converted to the yellowproduct more rapidly. A small amount of it is extracted with benzene.Based on these observations a method has been devised for the photometricdetermination of hydroxyproline in amino acid mixtures.  b  y  g u e s  t   , onM ar  c h  8  ,2  0 1 2 www. j   b  c . or  gD  ownl   o a d  e d f  r  om   806 NINHYDRIN METHODEXPERIMENTAL Amino At%&Preliminary Observations-The reaction of alanine with ninhydrin undervarying conditions was first explored. The conclusion of Moore and Steinthat the optimum pH for the reaction was at 5 was confirmed. At roomtemperature the reaction was very slow and, on completion, fell far shortof giving the theoretical amount of color. The reaction was speeded upconsiderably in the presence of excess of alcohol, both rate and yieldincreasing progressively as the concentration of alcohol rose from 80 to 98per cent. Other water-miscible organic solvents gave similar results. Py-ridine was particularly effective (6), as was a concentrated solution ofphenol in alcohol. Ultimately we chose an 80 per cent solution of phenolbuffered with pyridine as the most effective solvent.In the early experiments ascorbic acid was used as the reducing agent orhydrindantin itself was added to the reaction mixture.In the search for amore stable and soluble reducing agent we observed that potassium cyanideled to the formation of hydrindantin when added to a solution of ninhydrin,This was inferred from the observation that a red color was formed inborate buffers and a blue color in dilute sodium hydroxide.The spectraof these two colors were indistinguishable from those of hydrindantin in thesame two solvents.The stability of DYDA in the phenol-pyridine system was examined.In the absence of ninhydrin the color faded rapidly at loo”, but in thepresence of ninhydrin there was no detectable loss of color after heating for20 minutes. The explanation of this protective effect of ninhydrin is notknown.On the basis of the above observations the procedure described belowwas devised and tested on seventeen amino acids and a few peptides.Recommended Method for Primary Amino AcidsReagents-I. Ninhydrin solution. 500 mg. of ninhydrin are dissolved in 10 ml. ofabsolute alcohol.2. 80 per cent phenol solution. 80 gm. of reagent grade phenol aredissolved in 20 ml. of absolute alcohol, with gentle heating. The solutionis shaken with 1 gm. of Permutit for about 20 minutes to remove traces ofammonia and then decanted.3. KCN-pyridine reagent.2 ml. of a 0.01 M solution of KCN are dilutedto 100 ml. with ammonia-free pyridine, prepared by shaking 100 ml. ofpyridine with 1 gm. of Permutit for about 20 minutes.4. 60 per cent alcohol (by volume).  b  y  g u e s  t   , onM ar  c h  8  ,2  0 1 2 www. j   b  c . or  gD  ownl   o a d  e d f  r  om 
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