Charles R. Dawson

Comparison of the contact allergenicity of the four pentadecylcatechols derived from poison ivy urushiol in human subjects

Abstract Urushiol, the component of poison ivy sap which induces contact dermatitis, has been fractionated into its four constituent catechols. These catechols differ in degree of unsaturation of the pentadecyl side chain, ranging from the fully saturated pentadecylcatechol (PDC) to the tri-olefin derivative. Twenty six volunteers with histories of Rhus dermatitis were studied. Serial dilutions containing 10 −2 , 10 −3 , and 10 −4 μM of PDC, mono-, di-, and tri-olefin were applied to the skin of the back, and responses were recorded on Days 2, 3, 4, and 7. Reactivity was recorded as the highest dilution causing a popular to vesicular lesions on Day 7. Cutaneous reactivity was greatest to the di- and tri-olefins, followed in decreasing order by mono-olefin and PDC. When volunteers were grouped according to whether their last episode of Rhus dermatitis was either 5 or fewer years ago or 6 or more years ago, more reactivity to each test catechol was demonstrated the former group. When volunteers were grouped according to age, 20 to 39 and 40 to 59 years, greater reactivity occurred in the older group. This greater reactivity was most likely related to more recent exposure to the antigen —7.3 versus 3.0 years, respectively.

ON THE NATURE OF COPPER IN ASCORBATE OXIDASE. I. THE VALENCE STATE OF COPPER IN THE DENATURED AND NATIVE ENZYME.

Abstract 1. 1. The valency state of Cu in the enzyme ascorbate oxidase ( l -ascorbate: O 2 oxidoreductase, EC 1.10.3.3) has been determined by liberating the copper from the enzyme in the presence of valence-specific Cu-chelating agents. The procedures were designed to prevent electronic changes in the copper valency as a result of enzyme denaturation during the assay process. 2. 2. By means of the two Cu(I)-specific reagents, cuproine and bathocuproine, it has been found that the prosthetic copper in ascorbate oxidase exists in a mixed valency state, corresponding to 25% Cu(I) and 75% Cu(II). This ratio of 1:3 corresponds to 2 atoms of Cu(I) and 6 atoms of Cu(II) per enzyme molecule. 3. 3. This same ratio was found for the mixed valency state in the denatured enzyme when the Cu(I) reagent, bathocuproine, and the Cu(II) reagent, cuprizone, were used simultaneously. 4. 4. Neither bathocuproine nor cuprizone reacted directly with the native enzyme at physiological pH 7.2.

Assay of protein-quinone coupling involving compounds structurally related to the active principle of poison ivy

Abstract An experimental procedure, employing the techniques of column chromatography, has been developed by which the reactivity of waterinsoluble o -quinones toward protein can be evaluated semiquantitatively. This procedure has been used to study the effect of ring methylation on the ability of 3-pentadecyl- o -quinones to react to form protein conjugates. In this way it has been found that 3-pentadecyl- o -quinone is most reactive of all the compounds tested, whereas 4,5-dimethyl- and 4,5,6-trimethyl-3-pentadecyl- o -quinones, in which both of the normal sites for nucleophilic attack by the protein are blocked, do not react to any appreciable extent. The reactivity of all three monomethyl-3-pentadecyl- o -quinones has been found to be similar to that of the unblocked quinone, but the data for these compounds also indicate that there is some loss of reactivity resulting from steric interference by the lone methyl group in each case.

On the reaction inactivation of ascorbic acid oxidase.

1. 1. The reaction inactivation of the copper protein, ascorbic acid oxidase, has been reinvestigated and new evidence concerning the cause of the inactivation has been obtained. 2. 2. It has been found that the main products of the enzymic oxidation, i.e., dehydroascorbic acid and water, are not responsible for the inactivation phenomenon. 3. 3. The enzyme inactivation is dependent on the time required for the accumulation of a by-product of the reaction. This by-product appears to be hydrogen peroxide. 4. 4. Only a very small amount of the by-product develops during the typical reaction, apparently as the result of a secondary and low-rate catalytic reaction. 5. 5. Hydrogen peroxide is a very effective inactivating agent against ascorbic acid oxidase and only very small amounts are required to account for the experimentally observable enzyme inactivation.

ASCORBATE OXIDASE AND RELATED COPPER PROTEINS

Having developed significantly during the past 25 years, the field of molecular evolution has found its ultimate form of expression in protein biochemistry. While many areas of study unite to constitute the discipline of molecular evolution, proteins are believed to be the primary products of‘ the genetic process, and hcncc the evolution of proteins can be taken as the primary result of genetic mutation. It is on this basis that many analogies can be drawn with regard to the chemical nature of 2 or more similarly functioning proteins from different species. In discussing protein evolution, one must consider whether the proteins under study are the products of divergent or convergent evolution. Divergent evolution entails the mutation of one structure to different structures, while convergent evolution involves adapted changes of different and unrelated structures t o structures closely resembling one another. Thus, homologous proteins (probably arising from gene duplication) are considered to have similar genetic origins and result from divergent evolution, whilc analogous proteins are considered to have different genetic origins and to result from convergent evolution. More often than not, it is difficult to differentiate between analogous and homologous proteins. To unequivocally prove analogy or homology, a great deal of evidence, such as a knowledge of the proteins’ sources, functions, and structural properties, must be obtained. The usual problem arising i n such proofs is in obtaining suficient data. In general, howcvcr, proteins may be considered homologous if they have similar functional properties and many physiochemical characteristics in common and if there is a good probability that they maintain similar genetic origins. Of the studies completed on homologous proteins, most noteworthy are those of hemoglobin ’’ and cytochrome r.:%. ’ Numerous books and reviews 2 . 5 10 have been published that relate protein structure and function t o molecular evolution and genetic expression. In addition to the rather clearly hoiiiologous groups of proteins, e.g. hemoglobins, cytochronie c, albumins, there are many groups of proteins whose members share similar traits but for which no evolutionary link has yet becn established. One such group is the “blue” oxidases.

On the nature and mode of action of the copperprotein, tyrosinase: I. Exchange experiments with radioactive copper and the resting enzyme

Abstract 1. 1. The exchange reaction between functioning tyrosinase and radiocupric ions has been studied. 2. 2. In general, high catecholase enzymes incorporated much more radioactive copper during catalysis of the oxidation of o-dihydric phenols than of monophenols. 3. 3. High cresolase enzymes retained little radioactivity in all experiments. 4. 4. The copper exchange data support the suggestion of two distinct activity sites in tyrosinase; i.e. catecholase and cresolase activity centers. It appears that the copper at cresolase activity sites is non-exchangeable. The magnitude of exchange of the copper atoms at catecholase activity sites seems to depend on the number of o-dihydric phenol molecules oxidized. 5. 5. The catecholase sites of tyrosinase appear to be little or not at all involved in the oxidation of monophenols. 6. 6. On the basis of the exchange experiments it is suggested that the oxidation of a monophenol by tyrosinase may not proceed via an o-dihydric phenol.

[147] Ascorbic acid oxidase: Ascorbic Acid+12O2→Dehydroascorbic Acid+H2O

Publisher Summary This chapter discusses the determination of ascorbic acid oxidase. The assay method most generally used depends on the fact that, within a certain range of enzyme concentration, the rate of oxygen consumption during the oxidation of L-ascorbic acid is proportional to the amount of enzyme present. One unit of ascorbic acid oxidase activity is defined as that amount of enzyme that causes an initial rate of oxygen uptake of 10 μl. per minute. Specific activity is expressed as units per milligram of dry weight. The preparation of highly purified ascorbic acid oxidase is attempted from eleven different plants and found the yellow summer squash , Cucurbita pepo condensa , to be the most satisfactory source. Procedures developed have resulted in the preparation of solutions of enzyme that are homogeneous electrophoretically and in the ultracentrifuge. Ascorbic acid oxidase from squash is a blue protein having a molecular weight of 150,000, a copper content of 0.25%, and properties of a globulin. Maxima in its absorption spectra appear at 288 and 605 mμ. Homogeneous ascorbic acid oxidase is shown to possess a specific activity of 2000 units/mg. and 750 units/γ of copper. Purified ascorbic acid oxidase shows marked specificity for L-ascorbic acid. D-Ascorbic acid and a number of other ene-diols similar in structure to ascorbic acid are also oxidized although much more slowly.

Substrate specificity of ascorbate oxidase.

Abstract Ascorbate oxidase oxidizes leuco 2, 6-dichloroindophenol to the blue quinoid dye and produces spectral changes in the UV spectra of certain substituted polyhydric and amino phenols at pH 5.7. The new peaks produced by the addition of enzyme to the dichlorohydroquinones (2,5 and 2,6) and hydroxyhydroquinone correspond to the respective p-quinones of these substrates. At pH 5.7, the enzyme does not oxidize hydroquinone, barely oxidizes chlorohydroquinone, but oxidizes 2,6- and 2,5-dichlorohydroquinone and hydroxyhydroquinone at a rate about 1 12 that of ascorbic acid, with the uptake of one gram atom of oxygen per mole of substrate. A correlation has been found between the concentration of anion present in solution at pH 5.7 and the rate of oxidation of compounds of the hydroquinone series by the enzyme. The results indicate that an anionic form of the substrate is an important requirement of the enzyme specificity.

The exchangeability of copper at active sites in ascorbic acid oxidase

Abstract The exchange reaction between the copper protein, ascorbic acid oxidase and radioactive Cu 64 (cupric) ions has been reinvestigated under a variety of conditions. It has been confirmed that the exchange with the nonfunctioning (“resting”) enzyme is very much less than that, observed with the functioning enzyme and is dependent on the specific activity. The very rapid exchange observed with the functioning enzyme increases with the amount of substrate oxidized, and is a consequence of the catalytic mechanism rather than the processes by which the enzyme becomes inactivated. It has been shown that the exchange occurs at copper sites involved in the enzyme activity and such sites remain active following the exchange. The results are discussed in terms of different types of copper and copper bondings in the enzyme.

ON THE NATURE OF COPPER IN ASCORBATE OXIDASE. II. THE ROLE OF PROSTHETIC COPPER IN THE ENZYME'S FUNCTION.

1. 1. The respective roles of prosthetic Cu(I) and Cu(II) in the function of ascorbate oxidase (l-ascorbate: O2 oxidoreductase, EC 1.10.3.3) have been examined with respect to the blue color, activity and inactivation of the enzyme. 2. 2. It has been found that the Cu(I) fraction, representing approx. 25% of the total copper in the native enzyme, does not participate in the enzymatic activity or contribute to the blue color. That is to say, the complexing of that fraction of the protein copper by a Cu(I)-specific chelating agent does not affect the activity or the blue color. 3. 3. The Cu(I) fraction existing in the native enzyme can not be complexed directly with the chelating agent, except when the enzyme is functioning. It is concluded, therefore, that a reversible structural modification in the conformation of the protein moiety occurs during the catalytic cycle, thereby making this non-functional Cu(I) available to the reagent. Furthermore, the configuration of the protein and the binding of its functional Cu(II) fraction, are such that the continually generated Cu(I) component of the reversible Cu(II) ⇋ Cu(I) catalytic cycle is at no time available for complexing with the chelating agent. 4. 4. It has been found that the non-functional Cu(I) fraction of the enzyme is responsible for the production of the H2O2 that results in the characteristics inactivation of the enzyme during aerobic function. 5. 5. It was shown that small amounts of H2O2 have no inactivating effect on the resting enzyme but are strikingly effective on the functioning enzyme. The enzyme thus inactivated, loses its blue chromophore, but retains its copper. It is suggested that this H2O2 effect may involve directly the functional Cu(I) sites or the irreversible oxidation of some critical functional group exposed during the modification in structure of the protein moiety during catalysis.