Specific Enzymatic Determination of Some Alpha-Amino Acids by an Automatic Spectrophotometric Reaction Rate Method H. V. MALMSTADT and T. P. HADJIIOANNOU Deparfment of Chemistry and Chemical Engineering, Universify of Illinois, Urbana, 111.
b An automatic spectrophotometric reaction rate method is described for the selective enzymatic determination of about lo-’ equivalents of some L-a-amino acids. The method is based on the coupled enzyme reaction in which oxidative deamination of certain L-a-amino acids is specifically catalyzed b y L-amino acid oxidase to form hydrogen peroxide, which reacts with o-dianisidine in the presence of horseradish peroxidase to form a colored reaction product which has its absorption maximum a t 440 mp. The time required for the reaction to produce a small fixed amount of colored product, and therefore for the absorbance to change b y a preselected amount (about 0.06 unit), is measured automatically and related directly to the initial amino acid concentration. Small volumes (about 4 ml.) of nine different L-a-amino acid solutions were determined in the concentration range of 4 to 50 p.p.m. with relative errors of about 2%. Measurement times vary from a few seconds to about 2 minutes.
s
have been used for the determination of amino acids. Loy and Wright (3) described a microbiological tube assay for the analysis of L-amino acids. The ninhydrin reaction h a s been successfully applied to the continuous determination of amino acids in ion exchange column effluents (7’). Blaedel and Todd ( I ) developed an indirect method for continuous polarographic analysis of alpha-amino acids. A selective enzymatic method for determining the primary amino group in certain D-a-amino acids has been described by M a and Breyer (4). I n this paper, a specific enzymatic reaction rate method for the determination of some L-a-amino acids is presented. The method is based on the coupled enzyme reaction, in which oxidative deamination of certain L-aamino acids is specifically catalyzed b y L-amino acid oxidase to form hydrogen peroxide. The hydrogen peroxide reacts immediately with o-dianisidine in the presence of horseradish peroxidase EVERAL METHODS
14
ANALYTICAL CHEMISTRY
to form a colored reaction product, A few seconds after the start of the reaction and under selected conditions, the rate of formation of the colored reaction product is proportional to the initial amino acid concentration. The time, t, required for the reaction to produce a small fixed amount of colored product, and therefore for the absorbance to change by a preselected amount (about 0.06 unit), is measured automatically and related directly to the initial amino acid concentration. Micro amounts of several amino acids in the range of about 20 to 200 pg. were determined with reproducibility and relative errors of about 2q7& The measured samples contained amino acids at concentrations of about 4 to 50 p.p.m., in about 4 ml. of solution. The detailed mechanism of the selective oxidation of amino acids in the presence of amino acid oxidase remains to be elucidated, but the over-all process may be expressed b y Equation 1.
acid concentration. For the majority of the analyzed amino acids, the reciprocal times us. concentration curves pass through zero. I n these cases, a time interval measured for one standard should be sufficient to construct the standard curve. Ho\yever, a t very low amino acid concentration, a relatively large percentage of amino acid has reacted when the measurement is conipleted and the rate of the reaction is not proportional to the initial amino acid concentration. Also, a t high concentrations the rate is no longer proportional to the initial concentration of the amino acid because of partial saturation of the enzyme (amino acid oxidase) with substrate. I n other words, the reaction of a high percentage of amino acid at low concentrations would cause the extrapolated curve to pass below the origin; likewise, extrapolation from high concentrations, IF here substrate saturation has begun, would cause the extrapolated curve to pass
By adjusting conditions the rate of reaction is made pseudo first-order with respect to the amino acid. The hydrogen peroxide formed reacts immediately with o-dianisidine in the presence of horseradish peroxidase to form a colored product which has a maximum absorbance a t 440 mp. The rate of this latter reaction is considered instantaneous compared to the amino acid oxidation summarized b y Equation 1. The rate of formation of the colored reaction product, and the resulting rate of change of absorbance of the solution is, therefore, proportional to the initial amino acid concentration, provided that the measurements are completed before a significant amount of amino acid has reacted. As previously shown ( 5 ) , the reciprocals of the time intervals required to produce a preset amount of reaction product should be proportional to the initial concentration of the sought-for species under the desired conditions. The standard curves obtained when these reciprocal times are plotted against concentration are straight lines over a few fold change of amino
above the origin. Substrate concentrations larger than 0.01Ji are inhibitory. Therefore, standard curves are established b y using several standards. The standard curve is rapidly established from about 3 to 4 standards because each standard requires an average of about 1 minute to obtain the readout value. More accurate results are obtained when the unknown falls within the concentration range of the tested standards. If a series of unknowns is to be analyzed, standards are run occasionally to check the standard curve. Basic considerations concerning the effect upon the rate of the reaction of such variables as pH, temperature, and enzyme concentration are similar to those presented for the determination of glucose and alcohol in blood (5, 6). The reaction takes place in a thermostated cell a t 25’ =t0.1 C. A composite reagent containing amino acid oxidase and horseradish peroxidase in a buffered solution is unstable at this temperature, so the two reagents are added separately. The amino acid oxidase solution is kept in an ice bath
during the measurements and is drawn into a syringe only a few seconds before its injection into the reaction cell. This solution does not lose significant activity for a few days if refrigerated. Hon e\-er, the amino acid oxidase solution becomes cloudy upon standing and the preparation of a fresh solution daily is recommended. hlore precise results are obtained n-hen the amino acid oxidase solution is prepared 1 hour prior t o the start of a series of determinations. ‘1’0 preTent the enzyme from inacti1 :ition the amino acid oaidase is dissol.c-rtl in phoy~liate buffer which is 0.01JI in chloride ions. The univalent anions are effective in stabilizing the enzyme; they are not required for activity ( 2 ) . L-amino acid oxidase cataIyzes the o\idatii e deamination of the L-amino acids only and does not attack the r)-xniino scids. Because L-amino acid oxidase is sharply differential in its action, this enzyme can be used for the dfterniination of a-amino acids of the L-configuration, of L-amino acids in racemates, and of small amounts of r>-a-amino acids in preparations of
D-a-amino acids. A hydrolyzate of a protein or peptide cannot be analyzed because the enzyme does not attack all the L-amino acids. Isoleucine, a-aminon-butyric acid, citrulline, histidine, leucine, methionine, norleucine, norvaline, phenylalanine, tryptophan, and tyrosine are oxidized in the presence of the enzyme t h a t was used, but the method cannot be used for the determination of histidine and tyrosine because the rate of the reaction is decreased in the presence of the D-configurations of these two amino acids. Benzoic, mandelic, salicyclic, and iodoacetic acids, sulfonamides, aromatic sulfonic acids, aliphatic a-amino-sulfonic acids, and the carbonyl reagents all inhibit in a competitive manner ( 8 ) . Alanine, arginine, aspartic acid, cystein, glutamic acid, proline, serine, threonine, and valine are inactive. After initiation of the reaction a minimum premeasurement time of 10 seconds is desirable t o ensure thorough mixing of the reagents. The premeasurement time can be controlled b y appropriate setting of the comparator zero adjust. For example, the zero
Table 1.
for Aqueous Solutions of Amino Acids
Automatic Reaction-Rate Results
Amino acid 1.-e-amino-n-butyric acid citrulline
ii~-Citrulline L-Isoleucine
2-10 2.5-10
u~-Isoleucine L-Leucine
2.5-6
DL-Leucine L-Methionine
1.5-3
IJL-hlethionine t-Norleucine
2-4
m-Sorleucine (synthetic) 1.-Iiorvaline
6-15
DL-Norvaline L-Phenylalanine
1-2
uL-Phenylalanine L-Tqptophan
Amino acid in 4.1 ml. sample,n pg. Taken Found 65.0 65.0 130.0 130.0 130.0 131.0 70.1 69.7 70.1 70.8 105.1 104.1 27.8 27.6 83.4 81.8 27.8 28.3 83.4 85.4 39.4 39.8 29.4 38.7 18.7 77.8 39.4 39.4 39.4 40.4 78.7 78.7 29.8 29.6 29.8 30.4 44.8 45.2 39.4 39.4 39.4 39.2 39.4 39.2 70.3 68.3 70.3 70.9 140.6 138.8 140.6 140.1 117.1 113.8 16.5 16.5 16.5 16.3 33.0 32.8 33.0 33.6 30.6 30.1 61.3 60.6 61.3 62.6 30.6 30.9 61.3 62.0 61.3 61.3 to that of the L-isomer.
Concentration JJ x 104 7.5-25
1.5-4
DL-Tryptophan
Concentrations of m-mixtures refer
Relative error
... +0:8 -0.6 +1.0 -1.0
-0.6 -1.9
+1.7 +2.4 +l.O
-1.8 -1.1
+i:5
...
-0.7 +2.0 +0.9
... -0.5
-0.5 -2.8 +0.9
-1.3 -0.4 -2.8
...
-1.2 -0.6
$1.8
-1.6
-1.1
+2.1 +1.0 +1.1
...
adjust was set most of the time at 5.25 if 0.100 ml. of enzyme solution was used, but higher settings were required when the enzyme solution became cloudy upon standing, or was highly colored at the start, or when 0.25 ml. of enzyme solution was used. Dilute amino acid solutions (10-4M) decompose upon standing. They should be prepared b y dilution from more concentrated solutions when needed. EXPERIMENTAL
Instrumentation. T h e lia
