Cofactor Investigation of Bovine Plasma Amine Oxidase

It has been reported that treatment of amine-diamine oxidase mixtures with sodium borohydride resulted in the formation of a stable substrate-pyridoxal phosphate complex. When this procedure was applied to the bovine plasma amine oxidase in the presence of ‘%-labeled substrate, the enzyme was inactivated and became radioactive. A pure radioactive product was isolated from acid hydrolyzates by ion exchange chromatography and preparative paper chromatography of the hydrolyzate. The compound was shown to be E-N-benzyllysine by mass spectrometry. This adduct was apparently a reduced intermediate in the catalytic pathway of substrate oxidation by the enzyme. Formation of a substrate-pyridoxal phosphate complex could not be confirmed.

When this procedure was applied to the bovine plasma amine oxidase in the presence of '%-labeled substrate, the enzyme was inactivated and became radioactive. A pure radioactive product was isolated from acid hydrolyzates by ion exchange chromatography and preparative paper chromatography of the hydrolyzate.
The compound was shown to be E-N-benzyllysine by mass spectrometry. This adduct was apparently a reduced intermediate in the catalytic pathway of substrate oxidation by the enzyme. Formation of a substrate-pyridoxal phosphate complex could not be confirmed.

Investigations
of diamine oxidases have led to the proposal that they are copper-pyridoxal phosphate enzymes (l-7). Evidence that the organic cofactor of the diamine oxidases is pyridoxal phosphate came from trapping experiments of the enzymesubstrate complex by NaBH4, for example, Buffoni performing trapping experiments with the pig plasma amine oxidase.
The product isolated was reported to be the reduced substratepyridoxal phosphate Schiff's base (6), Kumagai  The inhibition of the beef plasma amine oxidase by sodium borohydride was found to be dependent upon the pH of the reaction mixture.
However, no effect of NaBH4 in the absence of benzylamine was observed in the range from pH 6 to 9. At increasingly higher pH values beyond 9 activity losses were noted relative to the control sample.
At pH 10.5, approximately 50% activity remained following incubation of the enzyme with sodium borohydride alone. Therefore, the reactions of enzyme, benzylamine, and borohydride were conducted at pH 7.2 where pH effects upon the enzyme were not significant.
Rate of Enzyme Inactivation in Trapping Experiments-As shown in Fig. 1, the enzyme lost 82% of its initial activity after 3 hours while a control sample of enzyme treated in an identical manner except for the addition of NaBH4 lost only 8% of its activity.
In a separate experiment, the absorbance of the reaction mixture without NaBH4 was measured after 5 hours to estimate the amount of benzaldehyde formed. This quantity of benzaldehyde then was substituted for the amine substrate and incubated with another enzyme sample.
Sodium borohydride was added and the reaction was allowed to proceed for 5 hours. The enzyme, after passage through a column of Sephadex G-25 F, retained 99% of its initial enzyme activity.
Moles of W-Labeled Benzylamine Incorporated in Enzyme-The moles of substrate (or product) incorporated into the enzyme using 14C-labeled benzylamine was determined. The results, summarized in Table I, show that 1 to 2 moles of radioactive compound were incorporated per mole of enzyme. Hydrolysis of 14C-Labeled Enzyme-The purified labeled enzyme was hydrolyzed with 5.7 N HCl and an aliquot was applied to paper for chromatographic analysis.
The developed chromatogram showed a discrete radioactive compound with an RF of 0.53. The recovery of radioactivity at this stage was about 50% of the initial radioactivity.  and c, synthetic c-N-benzyllysine.
cedure." About 80 to 85% recovery of the applied radioactivity was obtained in this step.
The pooled radioactive fraction was further purified by Dowex 50-H+ column chromatography.
The column separated the lysine from the arginine, the label remaining with the lysine fraction.
Paper chromatography of the pooled radioactive fraction showed the presence of two ninhydrin-positive substances. The major component was lysine and the minor component contained the radioactivity.
In the final purification step, preparative paper chromatography led to the isolation of a pure radioactive compound.
The yield of radioactivity in this step was about 70%. Rechromatography of the eluted compound showed a single spot when the chromatogram was sprayed with ninhydrin and the radioactivity was associated with this compound.
Chemical Properties of Radioactive Product-The isolated compound was ninhydrin-positive, eluted at the lysine position in the automatic amino acid analyzer, and had an RF of 0.53 in the solvent system 1-butanol-HOAc-Hz0 (4 : 1:2). One possible structure for the compound was pyridoxylbenzylamine.
Therefore, this compound was synthesized and the absorption spectra of the radioactive compound were isolated from bovine plasma amine oxidase and authenic pyridoxylbenzylamine were determined (Fig. 2). The spectra of these two compounds are quite different, which rules out the possibility that the reduced Schiff's base of pyridoxal and benzylamine had been isolated.
Another possible structure, e-N-benzyllysine, also was synthesized as described above.
The spectra of these two compounds are very similar (Fig. 2).
Mass Spectra Analysis of Radioactive Compound-The pure radioactive compound which had been 0-methylated and trifluoroacetylated was analyzed in the mass spectrometer. The product gave only one peak on gas chromatography.
The mass Thus, the compound formed upon the addition of NaBH4 to a plasma amine oxidasebenzylamine mixture is e-N-benzyllysine and not pyridoxylbenzylamine. DISCUSSION The diamine oxidases are widely distributed in nature, being found in bacteria, fungi, plants, and animals (13). Mammalian diamine oxidases are found in the plasma and kidney.
Both types have been isolated in a highly purified crystalline form, making it possible to investigate the prosthetic groups of the enzyme (2, 4). Identification of the organic cofactor is difficult since it cannot be freed from the enzyme by conventional procedures such as acid or base treatment of the enzyme at 4" (1). There have been reports that the cofactor is pyridoxal phosphate.
Several have reported the isolation of the substratepyridoxal adduct from acid hydrolyzates of the NaBH1-reduced enzyme-substrate mixtures (4, 6, 7'~ using W-labeled substrates to provide a convenient and sensitive label of the product.
This trapping technique was applied here to bovine plasma amine oxidase.
Aerobic rather than anaerobic conditions (14) were chosen since they appeared to provide optimal conditions for trapping the cofactor (enzyme)-substrate complex. Sufficient amounts of enzyme and %-labeled substrate were used to make possible the isolation of a pure radioactive compound from the NaBH4 trapping experiment.
The spectrum, RF and chromatographic behavior of the product all indicated that the product was not the expected pyridoxal-benzylamine. The mass spectrum of the radioactive compound proves that the radioactive product isolated from the enzyme was E-Nbenzyllysine.
The question arises whether the production of e-N-benzyllysine in these experiments is peculiar to the bovine plasma amine oxidase or whether it was also formed in previously reported experiments with pig plasma amine oxidase (6), kidney histaminase (4), and the A. niger amine oxidase (7). All of these enzymes have been reported to contain pyridoxal phosphate as a cofactor. Consider first A. niger amine oxidase. The re-ported mechanism for the oxidation of substrate by this enzyme n-amino acid oxidase is not affected. Bovine plasma amine (15) is almost identical with that reported for the bovine plasma oxidase, however, is inactivated. amine oxidase (14). Thus, trapping the substrate on the enzyme with NaBH4 should yield the same product with both REFERENCES amine oxidases. We have examined the experimental evidence of Adachi et aE. (7) and find that the electrophoretic mobility and the fluorescence data of the isolated material from the trapping experiments do not agree with the properties of the synthetic e-N-ethyl-pyridoxal amine. Likewise, when the electrophoretogram of the product obtained from kidney histaminase and the synthetic N-histaminyl-pyridoxal amine are compared, they have similar electrophoretic mobilities but again their mobilities are not identical (4). Thus, the claim of these studies that the cofactor of the enzyme is pyridoxal phosphate cannot be accepted at the present time.
Two mechanisms may be proposed for the formation of e-Nbenzyllysine.
One is that the benzaldehyde reaction product subsequently forms a Schiff's base with a reactive lysine e-NH2 group of the enzyme.
This seems unlikely based on the present finding that the addition of NaBH4 to enzyme-benzaldehyde mixtures do not inhibit the enzvme.
The amount of benzalde-