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Von Seiten der bioanorganischen Chemie besteht ein großes Interesse daran, Modellsysteme zu entwickeln, welche die Funktionen nativer Enzyme nachahmen. Hierbei besteht das Ziel in der Synthese von niedermolekularen Katalysatoren, die unter moderaten Bedingungen eine hohe ökonomische und ökologische Effizienz aufweisen und gleichzeitig in der Lage sind, ein großes Substratspektrum mit hoher Stereoselektivität umzusetzen. Motiviert durch die Mannigfaltigkeit kupferkatalysierter Enzymreaktionen, wird im ersten Abschnitt dieser Arbeit ein Modellsystem der Galactose Oxidase hinsichtlich der Oxidations-Reaktion von Ethanol zu Acetaldehyd untersucht. Hierbei konnte der Phenoxylradikal-Komplex, die wichtigste reaktive Zwischenstufe des Katalysezyklus, auf unterschiedliche Weise in Lösung generiert und charakterisiert werden. Der zweite Teil dieser Arbeit konzentriert sich auf die Synthese und Untersuchung von fünf bidentaten Imidazol-basierten Modellsystemen für die Catechol Oxidase- und Tyrosinase-Reaktion. Zunächst werden die Catechol Oxidase-Aktivitäten der synthetisierten Modellsysteme unter Berücksichtigung der sterischen und elektronischen Einflüsse untersucht. Insbesondere die kinetischen Untersuchungen der Reaktion und die damit verbundene Detektion und Charakterisierung von Zwischenstufen erlauben erste Aussagen über den Mechanismus. Im Anschluss wird die weitaus komplexere Reaktionsfolge der Tyrosinase untersucht. Der Einsatz von unterschiedlichen niedermolekularen Katalysatoren und einer Vielfalt an monophenolischen Substraten liefert erste Hinweise für die notwendigen Eigenschaften des Ligandensystems. Die Detektion charakteristischer Intermediate während der Katalyse zeigt weitere Unterschiede der eingesetzten Kupfer(I)-Katalysatoren auf, was ein besseres Verständnis für die Entwicklung bzw. Verbesserung von Tyrosinase-Modellsystemen erlaubt. Die empirisch gewonnenen Daten werden hierbei durch DFT-Rechnungen begleitet. The fascinating but challenging mission of bioinorganic chemistry is to develop model systems that mimic the function of these native enzymes. This task involves synthesis of low molecular weight catalysts which simultaneously exhibit high economic and ecological efficiency, work under moderate conditions and are capable to convert a wide variety of substrates with high stereoselectivity. Due to the diversity of copper-catalyzed enzymatic reactions, the first part of this thesis deals with a model system of galactose oxidase catalytically mediating the reaction of ethanol to acetaldehyde. In this context the phenoxyl radical, which represents the most important reactive intermediate in the catalytic cycle, could be generated and investigated in solution in different ways. The second part of this work focuses on the synthesis and characterization of five bidentate imidazole-based model systems mimicking the copper enzymes catechol oxidase and tyrosinase. Initially, the catechol oxidase activity of the synthesized models systems was investigated whereby steric and electronic effects were taken into account. In particular, kinetic studies of the reaction and the associated detection and characterization of intermediates allowed determination of the mechanism. Afterwards, the more complicated tyrosinase reactivity was examined. Applying different low molecular weight copper(I) catalysts to a broad range of monophenols as employed substrates provided indications for the required properties of the ligand systems. The detection of characteristic intermediates during the catalysis revealed further differences of the developed copper catalysts, leading to a more detailed understanding of tyrosinase model systems. These empirical data were analyzed with the help of DFT calculations.

By means of the histochemical proof for phenoloxidase (PO, EC 1.14.18.1) 25 patients, seven with periodontitis, the occurrence of PO-positive cells (POC) in the mouth rinse (Sp), crevicular washing fluid (S) and the capillary blood (CB) was examined. An increase and activation of the eosinophil granulocytes (EOS) in probe material (Sp, S and CB) of periodontally diseased patients, as observed in diseased periodontal tissues by several investigators could not be found. Apparently, because the small number and high variability of EOS in the investigated materials the enzyme proof of these materials would not give more informations about this connection.

A comparative application of phenoloxidase-demonstration and Pappenheim staining allows a sure differentiation of phenoloxidase-positive cells to be made, which can be found in the peripheral blood with different activity and different pattern of response. While eosinophils, neurophils, monocytes and lymphocytes contain phenoloxidase, basophiles do not.

31 samples of potato varieties with slow, medium and fast rates of browning were studies. Characteristic enzyme patterns were obtained from polyacrylamide gel electrophoresic of the phenoloxidases of varieties with different discolouration rates. The differences lie mainly in the intensities of the enzyme bands. The qualitative determinaiton of the phenols showed no significant differences. Tyrosine, chlorogenic acid and caffeic acid produce coloured oxidation products; the characteristic colour gradations of in vivo browning were only observed in the presence of tyrosine. It is concluded that the same reactions take place during the discolouration of all the varieties.

Quantitative histochemical measurements of activities of enzymes were performed by temporal defined stages in development of Protophormia terraenovae. The effect of the specific insecticide of moult Duphar pH 60-38 activity of enzyme was evaluated. Activities of peroxidase and tyrosinase showed parallel oscillations, at which proof of both enzymes was restricted to the cuticle. The insects, treated with insecticide, showed a moulting, dependent on concentration. A transfer of maxima of activities is described.

Phenoloxidase containing cells (POZ) identified with histochemical techniques in the subcutaneous connective tissue showed a marked increase in their number in response to heteroplastic skin transplantation in the rat. The number of these cells reached a peak of the 6th day of transplantation. From this time on their number gradually decreased on the 14th as well. POZ never invaded the graft, they remained confined to the host's tissue. A similar proliferation of POZ was never observed in the vicinity of skin autografts. The possible role in immune reactions of these cells is discussed.

By means of histochemical methods it could be shown, that the human blood contains three different types of cells with phenoloxidase. The phenoloxidase activity of the large cells is methanol resistant. The cells are considered to be hematopoietic stem cells. The medium size cells are granulocytes, while the small cells are lymphocytes.

Small gut of the white rat was sectioned by the cryostat in slides of 10 microns and following effectors were investigated: KCN, sodium-diethyl-dithiocarbamate (DDC), NaF, sodium acetate, sodium pyruvate, 0.1 N HCl, 0.1 N NaOH, NaN3, and copper sulfate. Phenoloxidase is completely inhibited by DDC and HCl, incompletely by pyruvate and NaN3. Peroxidase also is completely inactivated by HCl and incompletely by NaN3. DDC has no effect on peroxidase, because a precipitate is formed. Neither of both enzyme activities is inhibited by KCN. In presence of KCN and copper sulfate an unspezific colouring of the tissue resulted in the phenoloxidase proof. Non of the investigated effectors is suitable to differentiate between phenoloxidase and peroxidase in histochemical investigations.

Using the histochemical method for phenoloxidase (PO) in albino rats, the development of carrageenan granuloma was investigated. It is shown that especially those cells containing the methanol resistent PO activity are the cells which were be found during the formation of the granuloma. These findings support our earlier suggestion that POC are involved in the defence reactions.

The use of the enzyme proof for the application of cell mediated immunological defence reactions was demonstrated with preparations of pus, xenogenic transplantations, delayed hypersensitivity, and the granuloma formation.

Using 10 microns thick cryostat-slides of spleen and gut from white rats the following substances (in 2 different concentrations) were tested as substrates for the enzyme phenoloxidase compared with dihydroxyphenylalanine: pyrogallic acid, resorcin, 2.4-dinitrophenol, tyraminhydrochloride, o-cresol, and epinephrine. Only pyrogallic acid and resorcin resulted in positive reaction. Resorcin showed a positive reaction only when the higher concentration is employed. The investigations show clearly that the detectable enzyme in gut, spleen, and oral cavity is a polyphenoloxidase, which occurs consequently not only in granulocytes.

It is shown that the light microscopic permanent detectable phenoloxidase activity, using dihydroxyphenylalanine as substrate, of the small intestine of white rats is localized in the most cases in eosinophilic granulocytes. The enzyme has been found as well in the cytoplasma as in the granules. The enzyme proof triggered in the granules the so called reverse effect. The results allow to conclude new aspects for the cell mediated immunological defense reaction.

According to recommended international standards edible fungi are blanched before salting and freezing. A study was conducted on the solution losses of Lactarius rufus due to blanching. Weight losses, changes of dry matter, raw fat, total nitrogen, amino nitrogen and ash contents as well as the pH value were determined when various methods of blanching were used. 3 min blanching at 95-100 degrees C was able to inactivate catalase and peroxydase while 6 min blanching was needed for inactivating polyphenoloxydase totally. After blanching there were 1/10 - 1/100 of spores left. During the 3 min blanching in water five times the quantity of mushrooms the losses of dry matter were about 10%; when doubling the quantity of blanching water the losses increased to 2-3 fold. The doubling of blanching time had no significant influence on the losses. The soluble dry matter content of blanched mushrooms was less than 50% of that of the fresh. Total nitrogen of fresh mushrooms was equal to that of the blanched but the amino nitrogen decreased to one tenth by blanching. The mineral element content of blanched mushrooms was about the half of that of the fresh. Blanching caused a slight decrease in the pH value. The necessity of the blanching of all edible fungi before freezing was discussed.

Oxidation of the o-diphenolic compounds, catechol, protocatechuic acid, caffeic acid, and chlorogenic acid by o-diphenol oxidase (EC 1.10.3.1) resulted in compounds with antiviral activity against herpes simplex virus types 1 and 2. In the system HSV-1/rabbit kidney primary cells, a 90 per cent plaque reduction was obtained by the oxidation product from 1 mug caffeic acid, 9 mug catechol, 13 mug chlorogenic acid, or 20 mug protocatechuic acid (per ml), respectively. In comparision to the basic compounds or those oxidized in the presence of bovine serum albumin, the inhibitory effect of oxidized o-diphenolic compounds free from albumin was higher. From the basic o-diphenolic compounds, only catechol and caffeic acid showed remarkable antiviral activities in the system investigated. Although there have not experiments on the mechanism of action yet been the compounds are supposed to act during the earliest phases of the HSV-multiplication, because o-diphenolic compounds had been found to be present in the virus-cell system only 90 minutes after infection. In this respect the substances were similar to those of humic acids which also inhibit the adsorption phase of herpes simplex virus. There were no essential differences between herpes simplex virus type 1 and type 2 in respect of their sensitivity to enzymatically oxidized o-diphenolic compounds.

In the connective tissue, diffusely scattered phenoloxidase containing cells (PO-cells) can be found by histochemical methods. The same cells were demonstrated in a recurrent malignant melanoma of the skin and in its lymph node metastases. The theory is proposed that PO-cells are the stem cells of malignant melanoma in contrary to the common view that melanoma derives from the melanocytes of the stratum cyclindricum of the epidermis. The occurrence and distribution of PO-cells in the rat give a good explanation for the appearance of metastasis within different organs and tissues.

Using the histochemical detection for PO the in vitro effect of several detergents was tested. Dimethylsulfoxide, Triton X 100, Sodium desoxycholate, Tween 20, and Cetyltrimethyl ammonium bromide were investigated to check their effects upon the morphology and enzymatic activity of human leucocytes. Cetyltrimethyl ammonium bromide seems to be most suitable for the disintegration of cells. It does not inhibit the PO-activity.

Phenoloxidase is a specific enzyme for defence cells. It has been histochemically investigated, if substances partly known as tyrosinase inhibitors inhibit the phenoloxidase activity too. The tested substances are: thiourea, sodium-hydrogensulfate, cysteine, 2,3-dimercapto-propanol, N,N-dimethyl-p-phenylendiamine, glutathione, and ascorbic acid. It was detected for all substances, except dimethylphenylendiamine, an inhibitory effect. An attack at the enzyme molecule could be demonstrated for thiourea and dimercaptopropanol. The possibility of enzyme inhibition by these sulphur containing substances is of clinical interest.

The eye of a 47 year old man with tyrosinase-positive oculocutaneous albinism, photophobia, nystagmus and visual acuity 0, 4-0, 5 was histologically examined after orbital exenteration for neoplasia. Histologic serial sections of the centre of the retina showed a continuous 6-8 cell-layer of ganglion cells, without any suggestion of a foveolar pit. The outer layers of the macular retina were altered secondarily by tumor-impression-folds; they were unremarkable at the periphery as were the acid mucopolysaccharides in the receptor region. Electron microscopy of the uvea and the retinal pigment epithelium showed a normal number of pigment granules but a deficiency of melanin, as well as structural anomalies. The absence of the foveolar pit and the decrease of visual acuity in tyrosinase-positive albinism is caused by definite morphologic alteration in the arrangement of ganglion cells in the macular region in the sense of a foveolar aplasia. The etiology is discussed. An identic anomaly has been described in aniridia, similar ones in other congenital ocular diseases.

Nude mice, who are deficient in T-lymphocytes, were investigated to test, if the T-lymphocytes belong to the POZ or not. The results did not solve the question. But it has been confirmed that the POZ of the tissues are haematopoetic stem cells. Regarding to the obtained PO evidence nude mice are deranged in the PO-cell system. This derangement might be the reason for the fact that these animals tolerate foreign tissues like tumours.

With the aid of the histochemical phenoloxidase (PO) assay, 12 substances were tested. On the one side they can act as substrates or as inhibitors of phenoloxidase and on the other side as immunogenic or immunosuppressive compounds. In particular the following substances were investigated: o-cresol, Levamisol, prednisolonebisuccinate, Bleocin, siliciumdioxide, 4-methylbrenzcatechol, +-catechine, thiomersal, D-penicillamine, hydrochinonemonomethylether, hydrochinonemonoethylether, and hydrochinonemonobenzylether. No changes in the phenoloxidase activity and in the morphological behaviour of the cells could be detected using Levamisol, prednisolonebisuccinate, siliciumdioxide, and Bleomycin, indicating an immunogenic stimulation or immunosuppressive effect. 4-methylbrenzcatechol is probably transformed into a soluble reaction product by phenoloxidase. It could not clearly demonstrated by the histochemical method, if this substance is used as a true substrate. +-catechine, thiomersal, and D-penicillamine cause strong to complete enzyme inhibition of the phenoloxidase containing cells (POC), which are located in the spleen and gut of the white rat. Hydrochinonemonoethylether, hydrochinonemethylether, and hydrochinonemonobenzylether cause an increased pigment fermentation, but they are not used as substrates for the PO.

Investigations performed with cryostat sections of the gut, spleen and lung of the rat have shown, that the substrate of PER H2O2 inhibits the PO activity in dependence of the concentration, and that obvious the PO substrate DOPA is no suitable H2-donator for the PER reaction. Therefore a false positive reaction by the PO proof is not to be expected. Copper sulfat does not influence the PO reaction, but it accelerates melanin formation, which causes an unspecific colouring of the tissue.

Ten potato varieties, with different rates of browning, were analyzed quantitatively for phenoloxidase, tyrosine, chlorogenic acid, caffeic acid, and for reducing substances (ascorbic acid). The rate of tyrosine turnover was calculated from the data. The fact that the further reactions of the primary oxidation products leading to browning only take place after complete oxidation of the reducing substances, was taken into account. This leads to the same classification of the varieties as does visual observation of the rate of discolouration. Thus a clear relationship between browning and potato constituents is demonstrated.

Investigations of tissues from a naevus pigmentosus and 5 malignant melanomas were carried out. In malignant melanomas, the number of phenoloxidase reactive cells and the quantity of preformed pigment are very variable. It is discussed whether the in vitro phenoloxidase reactive cells and the in vivo pigment formation can be interpreted as expression of a cell mediated immunological defence reaction.

Using preparations of leucocytes and lymphocytes the reaction of phenoloxidase containing cells (POZ) during the spontaneous rosette formation was investigated. Beside the hitherto known cells forms 3 new types of PO-positive lymphocytes were found. It was established that the large methanol resistent POZ appear in the preparations of lymphocytes, too. Regarding their composition, isolated lymphocytes represent therefore a changing population of cells. Although PO-positive cells take part in rosette forming, the results gave no clear-cut evidence whether T-lymphocytes also contain PO. The application of the PO-reaction opens new aspects in the haematological and immunological diagnostics.

By means of the phenoloxidase reaction it would be shown that the POZ appear in haemopoetic cell islands of the yolk sac being hatched 6 days and being hatched 11 days, they appear in embryonary tissues. Favoured target organs are the spleen and the bone marrow, though also at the same time can be seen some cells in the connective tissue of the embryo. POZ can also be found in the bursa of FABRICIUS. The participation of the POZ in immunological reactions is supposed.

Among the cells containing phenoloxidase (POZ) of the human blood there was an evidence of cell forms being morphologically similar to macrophages. Lymphocytes represented no uniform cell fraction. There is a variety of large cells with a different enzyme distribution and activity. All POZ cells can develop a cell form which can be considered as an eye cell. Hypothetically it is connected with the cell generation.