Determination of PQQ in quinoproteins with covalently bound cofactor and in PQQ‐derivatives

Abstract

Application of the so-called hexanol extraction procedure for PQQ determination, originally based on detachment of the cofactor from quinoproteins and conversion into PQQ-S,S-dihexyl ketal, leads in several cases to a number of products due to uncontrollable esterification. The present modified procedure, detaching the covalently bound cofactor and converting it into 4-hydroxy-5-hexoxy-pyrroloquinoline, was tested on a number of proteins. Only the expected product was obtained for the known quinoproteins, in a quantitative yield, as revealed by comparison with the values determined with the hydrazine method. Thus this independent method confirmed that bovine serum amine oxidase, porcine kidney diamine oxidase, dopamine /I-hydroxylase from bovine adrenal medulla, methylamine dehydrogenase from Thiobncillus versutus, glutamate decarboxylase from Escherichiu coli, and 3,4dihydroxyphenylalanine decarboxylase from pig kidney are really quinoproteins. Quantitative conversion was also achieved for condensation and addition products of PQQ (PQQ-acetone, PQQH,, PQQ-oxaxole, PQQ-dinitrophenylhydraxone, and PQQ-tryptophan). In view of this conversion and the fact that catalytic activity of PQQ is not required, the method seems suited to investigate the distribution of the cofactor in eukaryotes, especially in mammals where it is almost certain that PQQ occurs only in derivatized form. Finally, just like the hydrazine method, the hexanol extraction procedure seems unable to keep the structure of the cofactor as it exists in the active site, intact, as demonstrated for the pro-PQQ cofactor of methylamine dehydrogenase.

Soybean lipoxygenase‐1 is not a quinoprotein

“…Derivatization of protein-bound PQQ, protein solvolysis with acidic hexanol, ODS extraction and RP-HPLC were performed as described by Van der Meer et al [19] with 25 mg soybean lipoxygenase-1. 13S-hydroperoxo-octadecadienoate (13S-HPOD) was prepared by incubating 400 CM linoleic acid with 10 nM soybean lipoxygenase-1 in an oxygen-saturated 0.1 M borate buffer, pH 9.0, at 4°C.…”

“…Therefore, we conclude that no PQQ-DNPH adduct has been formed. as described in [19] was used as an alternate method to determine PQQ in soybean lipoxygenase-1. The UV spectrum of the complete reaction mixture was not significantly different from that of untreated enzyme.…”

“…Subsequent HPLC analysis of the ODS-extracted material did not reveal any compounds absorbing at 318 nm (cf. [19]). We used hexanol with the same product number from the same supplier as Van der Meer et al [19].…”

“…[19]). We used hexanol with the same product number from the same supplier as Van der Meer et al [19].…”

A chromophore in glutamate decar☐ylase has been wrongly identified as PQQ

“…In both these cases the method that gave a falsely positive indication of the presence of PQQ was based on extraction of the relevant enzymes after treatment with phenylhydrazine. The evidence that PQQ is a component of glutamate decarboxylase and other amino acid decarboxylases arose from treatment of the enzyme by a quite different and frequently used analytical procedure involving extraction with hexanol [19]. The fact that a falsely positive result was obtained with bacterial glutamate dccarboxylasc Icad~ to the conchlsion that, like the hydrazinc method, tl~e hcxanol cxtractiou pro+ ccdure is an unreliable indicator that PQQ is a component of any proteil~.…”