Hemocyanin characterization

Type III copper is characterized by antiferromagnetic coupling of a pair of copper atoms and strong absorbance at 330 nm. A single type III pair is found in hemocyanin, in which it is involved in O2 transport, and in tyrosinase, in which an oxygen is inserted into substrate. A pair of copper atoms is also found in the multi-copper ascorbate oxidase, but it is coupled to the type II copper in a trinuclear arrangement. 

Geyer, H., Wuhrer, M., Kurokawa, T. and Geyer, R. (2004) Characterization of keyhole limpet hemocyanin (KLH) glycans sharing a carbohydrate epitope with Schistosoma mansoni glycoconjugates. Micron 35, 105-106. 

Recently, we also found that the blue solution of oxyhemocyanin turns red-purple upon addition of a large quantity of ethyleneglycol. Characterization of the purple hemocyanin should contribute to the understanding of the structural aspects of native hemocyanin. 

A trans-n- 1,2-Peroxo Dicopper(II) Complex. Our own efforts have resulted in the structural and spectroscopic characterization of five types of copper-dioxygen complexes (6), distinguished on the basis of the ligands used for their synthesis and on their distinctive structures or physical properties. Thus, the manner in which hemocyanin binds 02 is not the only one possible, and it is of considerable interest to deduce the structures, along with associated spectroscopy and reactivity of a variety of types. Dioxygen can bind to dinuclear transition metals in a variety of structural modes, shown in Figure 2. As mentioned, mode C is present in oxy-Hc and Kitajima s model complex (Scheme 1), whereas we have structural and spectroscopic evidence for types A (30-32), B (33-35), and F (36-38) for peroxo 022- binding, and mode D (39, 40) in the case of hydroperoxo (OOH ) complexes. 

Dnring the late 1970s, Solomon and collaborators prepared and extensively characterized a series of derivatives leading to models of the copper active-site structure and it function that are snmmarized in several articles. " While decades ago the end-on coordination was favored when interpreting the spectra, now great effort is applied to interpreting the spectra on the basis of the results of the recently resolved X-ray stracture, " which clearly show that dioxygen is bound in a side-on coordination between the two copper atoms. Several other contributions in this book will focus this point see Copper Hemocyanin/Tyrosinase Models). 

While the very rigid and, with respect to the type of donor groups and their geometric disposition (two trans-disposed pyridine donors and two cis-oriented tertiary amines), enforced and inflexible geometry precludes an accurate structural and spectroscopic modeling of copper proteins, it was especially feature (3.) that lead to the isolation and characterization of novel model complexes with hemocyanine- and catechol oxidase activities properties (81, 192, 196, 213). In the latter case, it was possible to isolate and structurally characterize complexes with coordinated catechol model substrates with structural features, which have been proposed to be of relevance in the enzyme catalysis cycle, but have not been observed before in low molecular weight complexes (192, 213). 

Details about the spectroscopic characterization of the (p-rj rj -peroxo) dicopper(II) complexes are presented later. It has been noted that the dioxygen binding of tyrosinase is reversible as in the case of oxy-hemocyanin (Scheme i) 32,3s,36 fonn of the enzyme is converted into the... 

Biomimetic copper-dioxygen chemistry has advanced considerably since the first structurally-characterized copper-dioxgygen adduct. However, it has been difficult to simulate the room-temperature stability of hemocyanin in these model complexes due to the fact that unlike the enzyme active sites, these models usually do not possess protective environments which can help stabilize potentially reactive copper-dioxygen species. Recently, two room-temperature stable copper-dioxygen complexes have been synthesized which come closer to the goal of mimicking the dioxygen carrier hemocyanin. 

Maddaluno, J. and Fault, K.F. (1999) Mass spectrometric characterization of Limulus polyphemus hemocyanin. Biochem. Biophys. Res. Common.. 264,883-890. 

Early determinations of the amino acid content of different hemocyanins were performed by Roche and Jean (1934) and Roche and Morugue (1941). More recently the amino acid content of hemocyanins from Limubts polyphemus and Helix pomatia has been determined chro-matographically by Barron and Johnson (unpublished) and Moring-Claesson (1956), respectively. Their results are reported in Table VI. One can see that not only are the values found for the two hemocyanins very different, but also notable differences exist between the old and the new data. Before it is possible to establish whether or not hemocyanins can be characterized on the basis of their animo acid content, more investigations on many other hemocyanins are necessary. 

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