The interaction of amino acids with o-phthaldialdehyde: A kinetic study and spectrophotometric assay of the reaction product

ANALYTICAL

BIOCHEMISTRY

188- 195 (1980)

101,

The Interaction A Kinetic

VYTAS-J. Chemical

K. SVEDAS,

Department

of Amino Acids with o-Phthaldialdehyde: Study and Spectrophotometric Assay of the Reaction Product IGOR J. GALAEV,

IVAN

und A. N. Belozersky Luboratory Lomonosov State University, Lenin

L. BORISOV,

of Molecular Hills, Moscow

AND ILYA

V. BEREZIN

Biology and Bioorganic 117234, USSR

Chemistry,

Received March 8, 1979 A detailed study has been made of the kinetics of interaction between amino acids and esters of amino acids and o-phthaldialdehyde in the presence of mercaptoethanol. The reaction products have been characterized. A spectrophotometric method for quantitative analysis of all amino acids, except proline and hydroxyproline, has been developed. The possibility of determination of amino acid esters in mixtures containing free amino acids has been demonstrated. It is noted that determination of glycine and histidine with the help of o-phthaldialdehyde has certain specificities associated with faster, compared to other amino acids, degradation of their derivatives. Optimal conditions for quantitative analysis of amino acids in solutions of higher than 10m5M concentration are recommended. The reproducibility of the determination was ?2%.

It is only recently that o-phthaldialdehyde has come into use as an agent which gives fluorescing products when combined with compounds containing an amino group, such as histidine, histamine, and various peptides which contain an N-terminal histidine (l-8); arginine and agmatine (9); spermidine (10, 11); glutathione (12); 3-, 5, and 3,5-substituted indoles, such as tryptamine and serotonin (13,14); and hydrazine (15). This property of o-phthaldialdehyde is used to detect nanogram quantities of compounds. Such determinations are conducted in strong alkaline media and a labile product is formed that fluoresces in the region 400-500 nm (excitation in the range 330-360 nm) and is stabilized by acidification. Mercaptoethanol allows the scope of compounds that produce a fluorescing product with o-phthaldialdehyde to be expanded. The method then becomes applicable to very different primary amines, including amino acids (but not proline and cysteine) (16-21) and peptides with a free amino group (22-26). o-Phthaldi0003-2697/80/010188-08$02.00/O Copyright All rights

Q 1980 by Academic Press, Inc. of reproduction in any form reserved.

aldehyde can also be used for developing chromatograms as it gives a fluorescing spot. However, the fluorescence of the spot is unstable and disappears in a few hours (25), which is not the case with other developers, e.g., fluorescamine. It is possible that the use of other SH-containing compounds, for example, ethanethiol, instead of mercaptoethanol, can improve the stability of fluorescing products (27). With some amino acids (glycine, tryptophan, arginine) (28-32) and amines (taurine, tyramine) (33), o-phthaldialdehyde produces colored substances in strong acidic or organic media. The color largely depends on the nature of an organic solvent. The effect of coloration can be used for selective determination of tryptophan and, especially, glycine in paper chromatography (31). In the previous studies we examined some properties of the products of interaction between o-phthaldialdehyde and amino acids and esters of amino acids (34,35). We concluded that o-phthaldialdehyde can be em188

INTERACTION

OF AMINO

ACIDS

WJTH

o-PHTHALDJALDEHYDE

189

15-sample distribution disk, each sample being placed in two separate wells. The first and second wells in each sample position were filled by micropipet with 0.25 ml of a solution of an amino acid and reagent, respectively. Then the disk was placed into an analyzer module where the reagent and sample were moved by centrifugal force through the wells of the distribution disk into thermostatted cuvettes and blown by air bubbles to mix the solutions. After that the analyzer measured optical density at certain time intervals. The time interval between two measurements varied from 5 to 99 s; the delay before the first measurement was set at 10 to 999 s. All optical density measurements were conducted with reference to a doubly diluted standard reagent. The second-order rate constant of the reaction between an amino acid and oEXPERIMENTAL phthaldialdehyde was determined as follows. Rerrgents. o-Phthaldialdehyde was a The pseudo-first-order rate constant of the product of Koch-Light; mercaptoethanol reaction was derived from the optical density of Merck; norleucine, tyrosine, ornithine, vs time relationship by the method of Hugenlysine, arginine, serine, and hydroxyproline heim (37) with a lo- to 20-fold excess of of Reanal; a-aminobutyric acid, tryptophan, o-phthaldialdehyde over the amino acid. alanine, leucine, and glycine of Reachim; The bimolecular rate constant of the reaction methionine, norvaline, valine, aspartic acid, was calculated from the tangent representing asparagine, cysteine, histidine, and phenylthe relationship between the pseudo-firstalanine of Chimreactivecomplect; C-phenylorder rate constant and the o-phthaldialdeglycine of Sigma; and threonine of Chemapol. hyde concentration. Mathematical treatment The rest of the reagents and salts were of of the experimental data was performed by Soviet production, chemical, and analytical the least-squares method, taking into acgrade. L-Tryptophan ethyl ester was pre- count the statistical weights of the pseudopared as described in Ref. (36). first-order rate constant, when the bimolecA standard solution of the reagent was ular rate constants were determined. prepared as follows: to 30 ml of 0.1 M borate The decomposition rate constant of the buffer, pH 9.7, 0.5 ml of ethanol solution of condensation product ofo-phthaldialdehyde o-phthaldialdehyde (10 mg/ml) and 0.5 ml of was determined by the method of Hugenmercaptoethanol solution (0.5 ~01% in heim (37). ethanol) were added. The reagent remained The kinetic parameters of the reaction stable for 24 h. The alcohol solution of o- were computed by numerical integration of phthaldialdehyde was stored in a dark vessel the system of two differential equations (38). for a month at room temperature. The optical The theoretical curve which was obtained density of the solution at 340 nm was meas- as a result of the integration was compared ured by a GEMSAEC 16-cuvette high-speed with the experimental curve by the standard analyzer manufactured by Electro-Nucledeviation technique. All the computations onics. The analyzer is supplied with a Teflon were carried out in a PDP-8/E computer. ployed for quantitative spectrophotometric assay of these compounds. The purpose of this work was a detailed kinetic analysis of the reaction betwen o-phthaldialdehyde and amino acids in the presence of mercaptoethanol and a quantitative description of the process. It was found that for determination of some amino acids (glycine, histidine), fast degradation of the compound should be taken into consideration. The data obtained have allowed optimal conditions for spectrophotometric determination of almost all amino acids (with the exception of proline and hydroxyproline) to be elaborated. The experimental data are interpreted in terms of the present-day views on the mechanism of the interaction between o-phthaldialdehyde and amines in the presence of SH-containing compounds.

190

SVEDAS

I

0.1

/\ 320

340

360

300

400

WAVELENGTH (nm) FIG. 1. The difference uv spectrum between the product of the valine reaction with o-phthaldialdehyde and doubly diluted standard reagent solution. Valine concentration

2 x

1O-4 M.

The difference uv spectrum for the solution of o-phthaldialdehyde and an amino acid, on the one hand, and the solution of the reagent, on the other, was measured in a Hitachi 124 double-beam spectrophotometer. All the experiments, including the kinetic studies, were conducted in a thermostatted cell at 25 + O.I”C. RESULTS

AND DISCUSSION

As was shown by us previously (34,35), interaction of o-phthaldialdehyde with amino acids in the presence of mercaptoethanol’ gives a product which can be detected spectrophotometrically by a characteristic absorption maximum at 340 nm (Fig. 1). It is obvious that the spectrophotometric amino acid assay with the use ofo-phthaldialdehyde requires a kinetic analysis of the reaction. The interaction between amino acids and o-phthaldialdehyde is very effective even in diluted solutions at room temperature and usually the reaction is completed within a few minutes (Fig. 2). The recorded optical density of the product solution is directly proportional to the amino acid concentration, and this makes a quantitative analysis pos’ Further on we shall not mention mercaptoethanol when speaking about the reactions between a-phthaldialdehyde and amino acids or derivatives.

ET

AL.

sible (Fig. 3). Direct proportionality is retained up to an amino acid concentration of (2-4) x lop4 M. Then the linearity of the optical density dependence on the amino acid concentration is interrupted, and it attains its maximum level when the amino acid concentration becomes commensurable with the concentration of o-phthaldialdehyde (Fig. 4). Therefore, this method of analysis allows determination of amino acid solutions of concentrations from (l-2) x lo+’ to (2-4) X It4 M. For elaborating optimal conditions for amino acid analysis by the o-phthaldialdehyde method, a detailed study is needed of the dependence of the reaction rate and product properties on o-phthaldialdehyde and mercaptoethanol concentrations, and on the pH value. We shall outline two groups of questions for study. The first comprises the relationship between the reaction rate of o-phthaldialdehyde and amino acids, or their derivatives, on the one hand, and other factors, such as initial substance concentrations, pH, etc., on the other. The second group deals with the relationship between the reaction rate and the extinction coefficient of the product. The recent works on the mechanism of interaction of amines with o-phthaldialdehyde (27,39) have made possible a comprehensive kinetic description of the reaction. In accordance with the proposed mecha-

FIG. during hyde;

2. The dependence of optical the reaction of isoleucine with 340

nm:

isoleucine

concentration

density vs time o-phthaldialde8 X lo-”

M.

INTERACTION

OF AMINO

ACIDS

WITH

191

o-PHTHALDIALDEHYDE

k,

A+B-+C-+P

k,

k, kz A+B+C-+D-+P

J

FIG. 3. The dependence of maximum optical density (340 nml vs concentration of (al lysine. (b) norleucine, (cl tryptophan, and (d) cysteine during the reaction with o-phthaldialdehyde.

nism of the reaction, a fluorescing l-alkylthio-2-alkyl-substituted isoindole (I) is formed at the first step, which is then subjected to a sulfur-oxygen rearrangement producing nonfluorescing 2,3-dihydro-IH-isoindole-lone (II). S-CH,-CH,-OH /

/

\ (r:“;

\

kz

IV

where A is a-phthaldialdehyde, B is an amino acid, C and D are spectrophotometritally detectable products, P is a nonabsorbing final product, k, is the second-order rate constant for the formation of an intermediate, k2 is the first-order rate constant for the decomposition of an intermediate, and k, is the first-order rate constant of the C to D conversion. The first step of the interaction of o-pthaldialdehyde and amino acids and amino acid esters obeys the second-order kinetics. As detailed analysis of experimental data showed, during the second step, the absorption decrease follows the first-order kinetics schemes. Kinetic analysis of schemes III and IV gives the following systems of differential equations:

s N-R

I

Iii

=i ti

-R

II

The spectrophotometrically recorded compound is, apparently, also 1-alkylthio-2-substituted isoindole. The uv spectrum of this product (Fig. 1) agrees well with the spectrum of 2-aryl isoindole, both of which have maxima in the region 335-340 nm and an extinction coefficient of es40 = 4800 M-’ cm-’ (40). For comparison, the extinction coefficient of the product of the reaction between valine and o-phthaldialdehyde is l s40= 5600 r 100 M-’ cm-‘. The substitutes in position 1 do not significantly affect the pattern of the spectrum (41). The two simplest kinetic schemes were analyzed:* ’ Since mercaptoethanol does not affect the kinetics of the reaction (341 it has been omitted from the scheme. Note. however. that the presence of mercaptoethanol is indispensable for the product to be formed. In the absence of mercaptoethanol. ophthaldialdehyde reacts only with cysteine.

i

i

I

dx = k,(a,, - x)(b,, - x), dt

x=c+p, & dt

V

= k,(x - p);

0.2

CVALI GKI FIG. 4. The dependence of maximum optical density vs relation of valine to o-phthaldialdehyde concentration: 340 nm: 0.4 M borate buffer, pH 9.6: mercaptoethanol concentration 6 X IO+ M.

192

WEDASETAL.

I

O.lt f-----4

dx = k,(a, - x)(&l - x), dt x=c+d+p,

dc = k,(ao - x)(b, - x) - k3c, dt

VI

dp

b

= k3d, dt

where x is the fraction of an amino acid or o-phthaldialdehyde invotved in the reaction, a, and 6, are the initial concentrations ofo-phthaldialdehyde and of an amino acid, and is time. Small letters designate instant concentrations of the substances. For realization of this amino acid analysis it is important to know the ratio between the rates of production and decomposition of the product to be analyzed. It turned out that the stability of spectrophotometrically detectable products is different for different amino acids. Of particular interest are quantitative descriptions of the time dependence of the optical density for amino acids which produce relatively low stable compounds in the reactions with o-phthaldialdehyde. The time dependences of the optical density for glycine and histidine are shown in Fig. 5. A quantitative description of the data in terms of schemes III and IV (systems of differential equations V and VI, respectively) shows that the best agreement between the estimates and experiments is observed with scheme IV. When the results were described in terms of scheme IV, the possibility of different extinction coefficients for intermediates C and D was taken into account. It turned out, however, that the best fit of the calculated curve to the experimental data is observed when the coefficients for the compounds hardly differ at all (the difference was less than 10%). This similarity of the spectral properties of substances C and D is understandable because I-hydroxy2-substituted isoindole (D compound) could have been the product of conversion of C (1-alkylthio-2-substituted isoindole). As shown in Ref. (42), the substitutes in posi-

t

II 0

I 30

IO

TIM&N) FIG. 5. The dependence of optical density vs time (340 nm). The curves are calculated according to the system of equations VI (scheme IV). (a) 10-I M histidine: k, = 22 M-’ s-‘. ttr = 8 X IO-” s-l, A:, = 4 X IO-:’ s-‘; cc,40 = 4800 M-’ cm-‘. (b) 5 x IO-” M glycine: k, = 930 M-l S-l, x, = 43 x 10-5 s-1: ,k:, = 22 x IOF SC’: .qLI,, = 4800 M-’ cm-‘.

tion 1 of the isoindole ring affect the spectral properties of the compounds very little. Note, however, that in the case of 2-unsubstituted isoindoles such compounds usually exist in the form of isoindolenine (VII). If, however, the hydrogen at the nitrogen atom is substituted, isoindole is stabilized into VIII (42,43). It is logical to assume therefore that the intermediates for D could be compounds of the VIII type where X is a hydroxyl. X

[I i:Ic:

‘N

VII

It must be noted that rapid decomposition of a registered compound is only significant in the case of glycine and histidine. If, however, o-phthaldialdehyde reacts with other amino acids, the stability of the compounds is much higher (the optical density decrease does not exceed 5-7% for 30 min). Thus, it may be concluded that it is only with histidine and glycine that the o-phthaldialdehyde spectrophotometric amino acid analysis requires more careful selection of the conditions for measurement. The kinetic parameters for the interaction of almost all amino

INTERACTION

OF AMINO

ACIDS

acids and o-phthaldialdehyde are presented in Table 1. The pH dependence of the bimolecular rate constant for the o-phthaldialdehyde interaction with amino acids has a bell-shaped pattern with a maximum in the 9.7- 10.0 pH range (Fig. 6). The effectiveness of amino acid interaction with o-phthaldialdehyde is controlled by two ionogenic groups with pK, 9.5 and pK, 1 I .O. The decrease of the rate of reaction between o-phthaldialdehyde and amino acids with the decrease in the pH of the solution is probably associated with protonation of an amino group of an amino acid (pK,). It is difficult, however, to say on the basis of the available data why the reaction rate drops at pH higher than 10 (pK,). TABLE

I

THE DIFFERENCE MOLAR EXTINCTION COEFFICIENTS AT 340 nm (E& FOR THE REACTION PRODUCTS, BIMOLECULAR RATE CONSTANTS (k,) FOR THE INTERACTION OF AMINO ACIDS AND O-PHTHALDIALDEHYDE, AND MONOMOLECULAR CONSTANTS (k,) FOR DECOMPOSITION

OF SPECTROPHOTOMETRICALLY

COMPOUNDS

DETECTED

Amino

acid

Glycine Alanine Valine Norvaline Leucine Norleucine lsoleucine Phenylalanine C-Phenylglycine cY-Aminobutyric acid Lysine Ornithine Arginine Histidine Glutamic acid Cysteine Tryptophan Methionine Serine Tyrosine Threonine Aspartic acid Asparagine

(M-’

GO Cm-‘)

5000 -t 100

k, x IO” cs-‘1

A, (M-’ 930

5400 5600 5300

t 100 + 100 -c 100

40r 38C 88+

48002 6400 5000

100 + 200 f 200

S-‘)

2

40

+ 30 k IO

430

2

40

6 4

II?

3

24t

7

60 + 20 382 2 572 I

19? 21k 14+

4 6 6

38k 405

2 4

172 322

5 9

100

542 loo+-20

6

23k 26k

6 7

66OOk 200 5700 ? 200

5022 58 2

6

18k

80 k IO 25k 7 32t 4

5700 2 100 5300 + 100 5800 t 8800~200

5

4800 4900 1550 4700

2 100 t 100

222

64t

4 3

k 70 k 100

3452 382

4

5200 5500 5200

2 100 t 100 2 loo

8Ok 442 332

4 6 I

222

6

l9k

4

5200 4800 4600

+ 100

202 442 395

2 2

342

8

s r

100 100

I

WITH

193

o-PHTHALDIALDEHYDE

301

” 9 1 2 IOI

a

I

I

IO

9

I

/

II

PH FIG. 6. The pH dependence of the second-order constant of valine reaction with o-phthaldialdehyde.

rate

In addition to determining the optimal kinetic conditions for the reaction, it was important to elucidate the conditions ensuring the most sensitive analysis. The task was accomplished by finding the factors which mostly affect the extinction coefficient of registered compounds (see Table 1). The effect of mercaptoethanol on the extinction coefficient of an analyzed compound was studied by us previously (34). Let us just note that under the conditions that are most favorable for amino acid determination, mercaptoethanol concentration must be not less than (0.6- 1.O) x 10e3 M. As analysis of the pH dependence of the extinction coefficient of the colored product of the reaction between an amino acid and o-phthaldialdehyde showed, the spectrophotometric properties of the compound to be determined are due to the ionogenic group with pK 9.0 ? 0.2. This ionogenic group is presumably the nitrogen atom in the isoindole ring. It is informative to compare this pH dependence with that of the extinction coefficient of the product of interaction of o-phthaldialdehyde with an amino acid ester. As seen in Fig. 7, the spectral properties of the product depend on the ionogenic group with pK 6.1 2 0.1. In this case, too, the nitrogen of the isoindole ring seems to be the ionogenic group. Note that the difference in the pK of these products (approximately 3 pH units) for an amino acid and an amino acid ester agrees well

194

SVEDAS

with the pK difference of amino groups of initial compounds (approximately 2.5 pH units) and is determined by different basicity of the nitrogen atom. This effect can be used for quantitative determination of amino acid esters in the presence of free amino acids. Indeed, when determining spectrophotometrically the product of the reaction between a mixture of an amino acid ester and an amino acid and o-phthaldialdehyde at pH 6-8, we mostly observe the product of o-phthaldialdehyde reaction with the ester. The differences in the pH profiles of the reaction rates between o-phthaldialdehyde and an amino acid ester or a free amino acid will contribute to the accuracy of amino acid ester analysis. In the 6 to 8 pH range, the majority of amino acid esters is in a deprotonated form, whereas practically all amino groups of all amino acids are protonated. This is the reason for the different rates of reaction between these compounds and o-phthaldialdehyde. The potentials of such an approach were explored in a kinetic study of enzymatic hydrolysis of tryptophan ester (35). Thus, on the basis of the above data, the optimal conditions for spectrophotometric determination of amino acids by means of o-phthaldialdehyde can be formulated as follows: the pH of the solution under analysis must be 9.7-10.0; the mercaptoethanol concentration must be higher than (0.6- 1.O) x 1O-3 M; and the o-phthaldialdehyde concentration must be two- to threefold higher than that of the amino acid. The optimal time of the optical density measurement (reagents are incubated at 25°C) is 3 to 5 min after mixing. It is especially important to choose the proper time of measurement for such amino acids as glycine and histidine. The sensitivity of the technique is practically the same for all amino acids (see Table 1). o-Phthaldialdehyde may also be used for quantitative spectrophotometric determination of amino acid esters. Moreover, under certain conditions (pH 6-8) esters of amino

ET AL.

5000 ig 5’ zB3000 w” -i/-

1000

6

6 p;l

FIG. 7. The pH dependence of the difference molar extinction coefficient (340 nm) for the product of the interaction between ethyl ester of L-tryptophan and o-phthaldialdehyde.

acids can be determined in the presence of free amino acids. It is interesting to compare the o-phthaldialdehyde method with other spectrophotometric methods of amino acid analysis. Compared to the routine ninhydrin method of quantitative analysis of amino acids (44), the proposed method is faster and less labor consuming, because boiling and sample storage after dilution are omitted. (The overall timesaving for one determination is about half an hour.) It is obvious that these advantages are especially important if a great number of determinations are required. The fluorescamine method of amino acid analysis (45-48) is relatively new. It must be noted that comparison of the sensitivities of this method and of the proposed method is difficult because in the former method amino acids are resolved in a column of an analyzer and then eluted as peaks. But it can be stated that the sensitivities of the two methods are roughly the same. The fluorescamine method detects up to 10 nmol (49), whereas the o-phthaldialdehyde method detects up to 5 nmol of a compound (a 0.25ml sample Of a concentration of 2 X lo-” M). However, o-phthaldialdehyde is more easily available and can be dissolved in water which is good for automatic dosing. Thus, in comparison with the routine spectrophotometric methods of quantitative

INTERACTION

OF AMINO

ACIDS

analysis of amino acids, the proposed o-phthaldialdehyde method has high sensitivity, simplicity, and rapidity. REFERENCES I.

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o-PHTHALDIALDEHYDE

195

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