Maria Teresa Graziani

Selective removal of type 2 copper from Rhus vernicifera laccase

Rhus vernicifera lactase contains, like fungal lactase, one Type 1, one Type 2 and two Type 3 copper ions [I] , which can all be removed by cyanide at pH 8.0 in a reversible process [2]. In the case of fungal lactase a method is described [3] which allows reversible removal of only the Type 2 copper. In view of the many analogies presented by these two proteins we have investigated the possibility of obtaining, also in the case of Rhus vemicifera lactase, selective removal of Type 2 copper. A method for the preparation of a protein depleted of Type 2 copper is reported in this communication.

A possible role for rhodanese: The formation of ‘labile’ sulfur from thiosulfate

Rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) is a well-known mitochondrial protein [ 1,2] . In spite of its widespread occurrence and abundance, its physiological role is very uncertain. In vitro it catalyzes the transport of sulfur from thiosulfate to a nucleophilic acceptor (cyanide, reduced lipoate) [3,4] by a double displacement reaction with the formation of an intermediate sulfur-enzyme complex [5,6] . Although some cyanide may be formed in vivo, this seems insufficient to explain the ubiquity and abundance of rhodanese. The possible significance of its action on reduced lipoate is also unclear. In the present paper a new role for rhodanese is outlined, namely the possibility that the enzyme may contribute to the formation of ‘labile sulfur’ in non-heme iron proteins from thiosulfate.

Identification of pantethinase in horse kidney extract

Purification of an oxygenase oxidizing cysteamine to hypotaurine has been recently reported from this laboratory [l] . Cysteamine, however, is not a common metabolite and we have sought a possible mechanism capable of producing the cysteamine used as substrate by this enzyme. Although a direct decarboxylation of cysteine has never been reported, it is known that cysteine is decarboxylated to cysteamine when it is bound to pantothenic acid in the course of the biosynthesis of coenzyme A [2]. Moreover pantetheine and phosphopantetheine are present in considerable amounts in the free or bound form in a number of tissues [3,4] . The enzymic cleavage of these compounds could represent, therefore, a source of free cysteamine. We have used pantethine as a representative substrate for checking the occurrence of a cysteamine (cystamine) producing enzyme in horse kidney. For convenience this enzyme will be referred to as pantethinase.

Electron spin echo spectroscopic studies of type 1 and type 2 copper in Rhus vernicifera laccase and in Cucurbita pepo medullosa ascorbate oxidase

Ascorbate oxidase and lactase are copper proteins that contain metal ions in at least 3 different chemical environments, 2 of which give rise to EPR [ 1,2]. In the oxidized state, both proteins are intensely blue and the color in each of them arises from a mononuclear CU(I1) site, designated type 1 [3] which is believed to be analogous to sites in the low Mr blue copper proteins, stellacyanin, azurin and plastocyanin. In the latter 3 proteins, the copper is ligated by cysteinyl sulfur (which gives rise to an RS-+ Cu(I1) charge transfer responsible for the intense blue color [4,5]) and by imidazole nitrogen [6-121. In plastocyanin and azurin, at a distance too far for charge transfer, there is a methionyl sulfur [ 11 ,121 which is absent in stellacyanin [ 13 ,141. A second copper site found in ascorbate oxidase and lactase, designated type 2, contains a metal ion in an environment which is not unlike that found in Cu(I1) peptide complexes [ 151. Except for the presence of an imidazole nitrogen in lactase [16], very little is known about the chemical nature of the type 2 binding site in either protein. A useful method for probing Cu(I1 jimidazole interactions in copper proteins and in models is provided by electron spin echo (ESE) spectroscopy [ 17,181. With this technique, one observes periodicities in the electron spin echo decay envelope which arise

Efficiency of nitrilotriacetate in the removal of type 2 copper from laccase and ascorbate oxidase

Abstract The powerful chelating agent nitrilotriacetate was found to be quite efficient in the removal of type 2 Cu, under reducing conditions, from the blue oxidases ascorbate oxidase and Rhus vernicifera laccase. While the effect of this substance on ascorbate oxidase was comparable to that of other reagents, as for instance EDTA, the effect on laccase was substantially larger than that of any other previously tested chelator, in particular EDTA which is by itself ineffective. This result confirms that the size of the chelator strongly affects its reactivity with laccase type 2 Cu, suggesting that the latter ion is more deeply buried in the protein matrix than the copper of ascorbate oxidase.

Spectrophotometric determination of thiols and disulfides with N-ethylmaleinimide

Abstract A simple and rapid spectrophotometric technique for the quantitation of thiols and disulfides has been described. The method involves the reaction with N -ethylmaleinimide before and after treatment with KCN, and is suitable for quantitative recovery of both thiols and disulfides from solutions in which they are present together.

The reaction of nitric oxide with Rhus vernicifera laccase

Among blue copper oxidases, laccases are the simplest, as each molecule of enzyme contains a single Type 1, Type 2 and Type 3 copper [ 1 ] . For this reason it seemed worthwhile to investigate the reaction of NO with a lactase, as this gas proved reactive with ceruloplasmin [2], which is a far more complex enzyme than lactase in terms of content and distribution of copper atoms. This communication deals with a set of experiments aimed to define the conditions of the reaction between NO and Rhus verniciferu lactase. The results obtained indicate that lactase and ceruloplasmin react with NO quite differently from each other.

Role of reduction potentials in copper abstraction from the trinuclear cluster of blue oxidases

Abstract The crucial point in the selective removal of the type 2 Cu from laccase and ascorbate oxidase by chelating agents was found to be the nature of the reducing agent employed in the reaction. No copper was lost when the reductant was either absent or too strong (ascorbate), namely when the trinuclear Cu cluster was respectively fully oxidized or fully reduced. From both proteins, the type 2 Cu was removed only in the presence of a mild reductant such as [Fe(CN) 6 ] 4− , the reduction potential of which could be varied by addition of [Fe(CN) 6 ] 3− and set at an intermediate value between that of type 3 and type 2 Cu ions. In this way the type 3 Cu ions were reduced with labilization of the oxidized type 2 Cu. Also, on prolonged incubation some type 3 Cu was removed from ascorbate oxidase. The applicability of the procedure to caeruloplasmin and fungal laccase is discussed.

Serum amine oxidase can specifically recognize and oxidize aminohexyl (AH) chains on AH-Sepharose support: single-step affinity immobilization1

Preparative affinity chromatography of bovine serum amine oxidase (SAO) on aminohexyl (AH)–Sepharose was often associated with an unexpected irreversible SAO retention on the support. This particular enzyme immobilization, occurring without coupling reagents, was supposed to be due to a SAO ability to: (i) recognize alkylamine groups of the support as macro‐molecularized substrate; (ii) catalyse their oxidation to the corresponding aldehydes, with release of NH3 and H2O2; and (iii) be immobilized on the activated support by a coupling between the nascent aldehyde groups and SAO free amine groups. This affinity immobilization procedure, with the self‐activation of the support, being mild, allows by simple incubation for 24 h, the enzyme immobilization with the retention of 80% from original specific activity of free SAO. Immobilized SAO on AH–Sepharose microcolumns, viewed as a continuous flow‐system reactor, was able to catalyse benzylamine oxidation for several weeks.

The 1H-NMR relaxation of Rhus laccase: Assignment to different types of copper

1H-NMR relaxation measurements of Rhus laccase showed that a portion of the relaxivity was specifically abolished by less than stoichiometric EDTA. Another portion of relaxivity was removed by addition of N3(-) to the EDTA saturated enzyme. This treatment or selective removal of the Type 2 Cu left a large residual paramagnetic relaxivity (1700 M-1s-1) which was assigned to the Type 1 Cu. It is concluded that only a portion of the laccase relaxivity can be assigned to the Type 1 Cu and that this copper type does not behave homogeneously: the two fractions have different relaxivity, 5200 and less than or equal to 2400 M-1s-1 respectively.