A color reaction between reduced pyridine nucleotides and o-aminobenzaldehyde of potential analytical use

ANALYTICAL

BIOCHEMISTRY

5,

255-269 (1963)

SHORT

A Color Reaction between and o-Aminobenzaldehyde

COMMUNICATIONS

Reduced Pyridine Nucleotides of Potential Analytical Use

The reaction of o-aminobenzaldehyde with certain pyrroline compounds to give characteristic orange-colored dihydroquinazolinium derivatives has been extensively studied by Schopf and collaborators (1). o-Aminobenzaldehyde has been used to trap and identify Al-pyrroline 5-carboxylate (2, 3) and Al-piperideine 6-carboxylate (4, 5) as intermediates in the biosynthesis of proline and lysine, respectively. We have recently described a cell-free yeast enzyme system that converts Naminoadipic acid to Al-piperideine 6-carboxylate in the presence of ATP, TPNH (DPNH), glutathione, and a divalent cation (Mn++) (5). One criterion used as evidence for this react,ion was the formation of color when o-aminobenzaldehyde was present. During one study of this reaction, a solution of the above cofactors plus o-aminobenzaldehyde in the absence of yeast enzyme was acidified and allowed to stand. Unexpectedly, this mixture also turned orange. Further investigation disclosed that the color resulted from a reaction of TPNH or DPNH with o-aminobenzaldehyde in acid solution. Figure 1 compares the visible spectra of the reaction product of DPNH plus o-aminobenzaldehyde with that of the dihydroquinazolinium derivative of Al-piperideine-2-carboxylic acid (t,he cyclized form of a-keto-E-aminocaproic acid). Color was obtained only with the reduced form of the pyridine nucleotides and was proportional to nucleotide concentration (Fig. 2A). This reaction was therefore applied to the assay of a pyridinenucleotide-dependent dehydrogenase enzyme system. A conventional spectrophotometric assay of glucose 6-phosphate dehydrogenase is compared with a simultaneous o-aminobenzaldehyde assay in Fig. 2B. If adequate controls are exercised to rule out other substances that may react with o-aminobenzaldehyde, the ability of DPNH and TPNH to form colored compounds with o-aminobenzaldehyde may have certain 255

256

I c

SHORT

0.0

COMMUNICATIONS

-

A'- PIPERIDEINE-Z-CARBOXYLIC

-----

OAB COMPLEX DPNH-OAB COMPLEX

ACID-

WAVELENGTH

FIG. 1. Absorption spectra of o-aminobenzaldehyde (OAB) adducts of Al-pipe& deine-2-carboxylic acid and DPNH. Spectral determinations were made with the Carey recording spectrophotometer (0.05 N HCl blank) of the following: a 0.05 N HCl solution, 0.5 mg/ml, of purified dihydroquinasolinium derivative of A’-piperideine P-carboxylate and OAB prepared as described elsewhere (5), and a solution (diluted 1: 2) in which 2.5 &I DPNH had been standing with 3.3 $rM OAB in 2 ml 0.05 N HCl for 1 hr. o-Aminobensaldehyde was purchased from K and K Laboratories, Inc.

analytical advantage over present methods. The o-aminobenzaldehyde assay is only about a third as sensitive as the direct spectrophotometric assay of reduced pyridine nucleotides and requires a 30-60 min interval for maximum development of color. On the other hand only a calorimeter is required and an alternative wavelength for the measurement of TPNH and DPNH is provided. These observations may be of particular use in those instances (e.g., teaching) in which it may be desired literally to visualize reactions in which these co-enzymes participate. ACKNOWLEDGMENT This work of Arthritis States Public

was supported in part by a Grant, A-3156, and Metabolic Diseases of the National Health Service.

from the Institutes

National Institutes of Health, United

SHORT

257

COMMUNICATIONS

L.”

DPNH-OAB COMPLEX FORMATION 2 0

% I

1.5-

l.O-

/

& z 2 8

DPNH

./ AL

O5-

/

2 Q :-!

0

0.5 AMOLES

IO PYRIDINE n

1.5 2.0 NUCLEOTIDE

i2: 5

.-GLUCOSE 6-PHOSPHATE DEHYDROGENASE ASSAY

, /

/ 0

I

I

I

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2

3

4

TIME

5

- MINUTES El

Fm. 2(A). Formation of DPNH-o-aminobenzaldehyde (OAB) adduct as a function of DPNH concentration. To a series of tubes containing 3.3 pM OAB in 0.5 ml 0.2 N HCl, increments of DPNH or DPN were added as indicated, the tubes brought to 2 ml with water, mixed, and let stand for 1 hr. Color formation was measured with a Spectronic 20 spectrophotometer at 450 mp. (B) Glucose g-phosphate dehydrogenase assay as followed by TPN reduction or formation of TPNH-OAB adduct. To 0.4 ml water in a 2-ml quartz cell having a l-cm light path was added 0.5 ml TPN (1.5 X IO-“M), 0.3 ml 0.04 M glycylglycine buffer (pH 7.5), 0.2 ml MgCL (0.1 Ml, 0.5 ml glucose B-phosphate (0.02 MI and 0.1 ml glucose bphosphate dehydrogenase (0.1 K unit, Schwartz). Optical density changes were recorded at 1-min intervals in a Beckman Model DU spectrophotometer at 340 rnp with results as indicated in the upper curve of B. To measure TPNH formed as the OAB complex, the components of the system just described were added in 5-fold greater quantities to a test tube, but, following addition of the enzyme and mixing, l&ml aliquots were removed at I-min intervals by pipetting directly into tubes containing 3.3 pM OAB in 0.5 ml 0.2 N HCl. After 1 hr, color formed in these tubes was measured spectrophotometrically at 450 rnp with results as shown in the lower curve of B.

258

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3. 4.

5.

SHORT COMMUNICATIONS

REFERENCES SCHOPF, C., KOMZAE, A., BRAUN, F., AND JACOBI, E., Ann. Chem. 559, 25 (1948). VOGEL, H. J., AND DAVIS, B. D., J. Am. Chem. Sot. 74, 109 (1952). STFCECKER, H. J., J. Biol. Chem. 225, 825 (1957). YURA, T., AND VOGEL, H. J., J. Biol. Chem. 234, 339 (1959). LARSON, R. L., SANDINE, W. D., AND BROQUIST, H. P., J. Biol. Chem. (in press).

R. L. LARSON H. P. BROQUIST Laboratory

of Biochemistry

Department of Dairy Science University of Illinois, Urbana Received

Fluorometric

November

d3, 1968

Assay

(Dimethylaminoguanine)

of 2-Dimethylamino-6-hydroxypurine in the

Presence

of Guanine

In a recent report (1) it was pointed out that 2-dimethylamino-6hydroxypurine (dimethylaminoguanine, DiMG) is a highly fluorescent compound, the quantum efficiency at pH 11 being several times greater than that of guanine. Furthermore, fluorescent characteristics of DiMG are different from those of guanine, the excitation and fluorescence maxima of DiMG being 290 and 368 rnp as compared to 275 and 350 rnp for guanine (corrected). Recent findings of DiMG as one of the minor components in sRNA (2,3) make desirable a specific fluorometric method for its assay. We have found that it is possible to measure small amounts of DiMG in the presence of large amounts of guanine by treating a mixture of the two purines with nitrous acid. By this procedure DiMG is unchanged whereas guanine is converted to xanthine, which is not fluorescent under the conditions employed. S-RNA samples were hydrolyzed in 1 N HCl at 1OO’C for 30 min. Aliquots of the hydrolyzate containing 10-15 pg of sRNA in 0.1 to 0.3 ml were transferred to glass-stoppered centrifuge tubes and the volume brought to 0.3 ml with 1 N HCl, when necessary. The tubes were then placed in a boiling water bath and 0.1 ml of a solution of sodium nitrite (1 mg/ml) was added. Additional (0.1 ml) aliquots of the sodium nitrite were added at 3-min intervals for a total of four additions. Three minut’es