Henry Tauber

Chemical Nature of Catalase

The chemical nature of catalase has been studied by a number of authors. The experiments of Zeile 1 and of Stern 2 indicate that the catalase molecule is probably a chromoprotein in which the hemin group is related to that of the natural blood pigments. Recently Agner 3 reported that if horse liver catalase is dialyzed against N/10 or N/100 HCl the enzyme splits into 2 inactive components, one of a low molecular weight which dialyses through the cellophane membrane and which is possibly hemin and another component which remains within the bag and which is a protein. If the 2 neutralized components are mixed, an active preparation is again obtained, according to Agner. Having the intention of making certain studies on the basis of Agners report, we first attempted to repeat his experiment. The technique is simple but we were not able to confirm his results. The only difference between his and our technique is in the species of animal from which the liver was derived. Horse liver, which Agner used, is practically unobtainable in our locality. We have, however, tried beef, rabbit, and rat liver, respectively. We followed the experiments of Agner in every detail, dialyzing the purified catalase against N/10 or N/100 HCl, for 10 to 48 hours. No splitting (inactivation) of the catalase could be obtained. With stronger HCl irreversible inactivation of the catalase took place, due to a marked decrease in pH inside the dialyzing bag.

Co-Carboxylase (Vitamin B1-Pyrophosphate) Content of Plants

Lohmann and Schuster 1 , 2 have shown that co-carboxylase is identical with the pyrophosphoric ester of vitamin B1. I have demonstrated recently 3 , 4 that synthetic vitamin B1 is readily phosphory-lated by the duodenal mucosa and by extensively washed dry yeast which had been freed of co-carboxylase. The enzymic synthesis of vitamin B1 -pyrophosphate occurs best at a slightly acid pH. Euler and Vestin, 5 and Kinnersley and Peters 6 independently and at the same time when I published my experiments have also shown that yeast converts vitamin B1 to co-carboxylase. The enzyme carboxylase has an important part in carbohydrate metabolism. It is widely distributed in nature and it is accompanied by its coenzyme. It occurred to me that a quantitative study of a series of plants for co-carboxylase and comparing these findings with the vitamin B1 content of the plant material may be of interest. The well ground plant tissues were extracted by boiling for 5 minutes with 3 to 5 times their weight of pH 6.239 phosphate (Sorensen). In 2 cc. of the clear nitrates co-carboxylase was determined according to Lohmann and Schuster. 1 In Column 1 of Table I, figures obtained for co-carboxylase are shown and in Column 2, figures for vitamin B1 content of these plant materials as obtained by biological assay are given. By comparing these figures with that which I have obtained for the enzymatically synthesized co-carboxylase (not completely phosphorylated) it may be seen that only a small part of the total vitamin B1 is present in these plants in the form of its pyrophosphoric ester. It should be noted, however, that there is no direct proportionality between CO2 formation and the amount of co-carboxylase added, especially when too large amounts of the co-enzyme are to be estimated. For this reason in the case of dry yeast only 0.3 cc. of boiled yeast juice equivalent to 50 mg. of dry yeast was employed for the test. The results show that phosphorylation of vitamin B1 by the mammalian organism is of vital importance as this co-enzyme plays an important rôle in the decarboxylation and dehydrogenation of pyruvic acid. 8 Furthermore animal tissues contain all of the vitamin B1 as the pyrophosphoric ester. 8

Color Test for Pentoses

One drop (0.05 cc.) of a pentose solution (1-arabinose, d-arabinose, xylose or ribose) containing 0.05 mg. or more of the sugar is placed in a test tube. 0.5 cc. of benzidine solution (1 gm. benzidine in 25 cc. glacial acetic acid. This solution keeps for 4 days) is added and the mixture is brought to vigorous boiling. The test tube is then put in cold water. Within a few seconds a very stable cherry red color develops. Glucose, fructose and galactose give yellow to brown colors with the benzidine solution. Even large amounts of hexoses, however, do not interfere with the test. There is only a slight delay in the formation of the red color (0.1 mg. of arabinose and one mg. of glucose). As little as 10 gamma of pentose may be detected. For instance urine specimens containing 0.1 mg. of arabinose per cc. gave a distinct red color when 0.1 cc. of urine was added to 0.5 cc. of benzidine solution. Normal and abnormal urinary constituents do not interfere with the test. Too large amounts of proteins may be removed from pathological urines by adding an equal volume of 10% trichlor acetic acid, mixing, and heating to 95°. The filtered urine may then be tested. When testing for pentose in urine, the urine (0.1 cc.) and benzidine solution (0.5 cc.) is brought to vigorous boiling. The mixture is cooled under tap water and 1 cc. of distilled water is added. In the presence of pentose a pink to red color is shown immediately, whereas if pentose is absent the mixture has a yellowish brown color. This color test is also given by vitamin B2 (riboflavin), by the yellow oxidation enzyme of Warburg and Christian, and by nucleic acids owing to the ribose content of all of these compounds. It is negative, however, with gum arabic since this pentosan is not hydrolyzed by the benzidine-acetic acid solution. The test is highly specific for pentoses.

Enzymes of Neisseria gonorrhoeae and other Neisseria.

Summary It was possible to demonstrate the existence of a potent reduced diphosphopyridine nucleotide oxidase in all Neisseria. This enzyme and alcohol dehydrogenase were increased in relative penicillin-resistant isolates of N. gonorrhoeae. Another potent enzyme found was diphosphopyridine nucleotide-linked lactic dehydrogenase. Some organisms contained much less LDH and DPNH oxidase than N. gonorrhoeae isolates. Reduced triphosphopyridine nucleotide oxidase was present only in small quantities in all organisms investigated. An acetate oxidizing enzyme system was present in large quantities in E. coli and S. aureus, in small quantities in N. sicca and TV. catarrhalis, and in traces in a few isolates of N. gonorrhoeae. The system was absent from N. meningitidis. For the above enzymes, the Thunberg technic with tetrazolium salt as the electron acceptor was employed. All Neisseria contained a cyanide sensitive, aerobic cysteine oxidase and a cysteine desulfhydrase. It is hoped that the present investigation will aid in identification and classification of Neisseria. These results were obtained with old stock cultures of N. gonorrhoeae. When a large number of fresh isolates was assayed only partial correlation between penicillin resistance and enzyme activity was observed.

Effect of cortisone, properdin and reserpine on Neisseria gonorrhoeae endotoxin activity.

Summary Cortisone significantly protected mice from the lethality of Neisseria gonorrhoeae endotoxin. Similar results were obtained with properdin, provided relatively large doses were employed. Reserpine, under certain conditions, significantly enhanced the toxicity of endotoxin.

Preparation and Some Properties of Neisseria gonorrhoeae Endotoxin.

Summary 1. An improved method for large scale cultivation of N. gonorrhoeae in an atmosphere of air has been described. 2. A rapid method for the preparation of a stable fraction containing dry, soluble gonococcus endotoxin and some of the properties of this product have been presented. This endotoxin appears to be a protein. 3. DNA equivalents in different gonococcus fractions do not parallel toxicity.

Stability of Vitamin C, and Absence of Ascorbic Acid Oxidase in Citrous Fruits and Milk

It has been shown recently that in certain plants such as Hubbard Squash and Summer Squash, a powerful enzyme is present which oxidizes rapidly vitamin C 1 2 although these plants contain almost none of this vitamin. Similar is the case with cucumbers. Statements have appeared more recently that “plant tissues which contain ascorbic acid apparently also contain an ascorbic acid oxidizing enzyme,’ and that the partial destruction of vitamin C in cows milk is also brought about by ascorbic acid oxidase. The present writer could not find ascorbic acid oxidase in mammalian tissue, 3 and Roe and Barnum 4 found in human and rat blood cells and plasma an enzyme which reduces the reversibly oxidized form of ascorbic acid, thus having just the opposite effect from the ascorbic acid oxidase of plant tissues. The aim of the present work was to find out whether the vitamin C content of juices of citrous fruits, which are excellent sources of the vitamin, is exposed to the destructive action of the ascorbic acid oxidase. In other words, whether these juices contain the oxidase. Table I shows that the vitamin C content of the juices of oranges, tangerines, lemons and grapefruits is not much affected when kept for 5 hours at 38° as compared to control samples which were placed in a refrigerator at 6° for the same amount of time. The slight decrease in reducing power does not necessarily indicate an irreversible oxidation. The results show, however, that there is no ascorbic oxidase in these fruit juices and that the vitamin keeps fairly well even at 38°. It is well known that the pH of these fruit juices is a stabilizing factor of vitamin C. Samples of orange juice which have been adjusted to pH 6.5 with ammonium hydroxide or CaCO3, however, kept equally well and no evidence of enzyme action could be noticed at this pH either.

Nature of polysaccharides obtained from endotoxins by hydroxylaminolysis.

Summary 1. There are marked differences between endotoxins prepared from 3 different species of gram negative bacteria. 2. The differences are retained after removal of the lipid component from the endotoxins by hydroxylaminolysis. Amino compounds are an integral part of both the endotoxin and polysaccharide molecules. 3. Molecular weights of the major components of the water soluble lipid-free materials are 200,000. We wish to thank Dr. Edgar Ribi of the Rocky Mountain Laboratory for determining the molecular weights.

Subtenolin. An Antibiotic from Bacillus subtilis. II. Isolation and Chemical Properties

Discussion and Summary It may be seen from Table I that subtenolin is quite different chemically from other antibiotics such as streptomycin, penicillin G, gramicidin, and bacitracin. Subtenolin, bacitracin, bacillin, subtilin, and eumycin are products of various strains of B. subtilis. Gramicidin 4 and bacitracin 5 appear to be peptides. Subtilin, which is extracted from the pellicles of a particular strain of B. subtilis, is of unknown chemical composition. 6 Eumycin is prepared by acid precipitation of the medium. 7 The chemical relationship between bacillin and subtenolin has not been ascertained since data pertaining to the chemical nature of bacillin is not available for comparison. 8 Subtenolin has a low molecular weight. Its chemical and physical properties indicate that it contains a resonating double bond, phenolic groups, a very active enolic group, and an aromatic aldehyde radical. This antibiotic gives typical color reactions such as the Molisch and enol reaction. These tests, and the indicator test shown in Table I, may be used in its identification. Although the antibiotic has not been obtained in a pure state, its chemical and antibiotic properties, particularly its stability in aqueous solution or when dry, make it an interesting object for further studies.

Isolation of a specific ascorbic acid (vitamin C) Oxidase.

An extract of Hubbard squash oxidizes both the synthetic and the natural ascorbic acid (vitamin C)∗ with great rapidity. This is due to an enzyme having an optimum pH of 5.83 to 5.96. It may be obtained and purified by extracting the squash (edible part) with twice its weight of 30% ethyl alcohol for 10 minutes. The centrifuged and filtered fluid is treated with an equal volume of acetone, which causes a yellow sticky substance to precipitate. This may be washed free of yellow pigment with acetone, dissolved in water and reprecipitated with acetone. A third precipitation yields a preparation, which after drying in vacuo over sulphuric acid has an activity 500 times that of the original extract. This preparation is water soluble and gives slight protein tests. Alcohol and saturated solutions of neutral salts, however, do not precipitate it. It is digested (inactivated) by trypsin. A polysaccharide accompanies the enzyme in the above precipitation. We have found no way of removing it thus far. This enzyme differs in various ways from the “hexoxidase” which v. Szent-Györgyi 1 discovered in cabbage leaves, e. g., the hexoxidase is precipitated by saturated (NH4)2SO4 solution; it oxidizes not more than about 25% of the substrate, whereas the enzyme of the squash oxidizes 100% very rapidly. Moreover, the kinetics of our preparation point to the presence of a single enzyme. Substances thus far tested, such as cysteine, tyrosine, glutathione, and phenols, are not affected. We suggest, therefore, that the enzyme responsible be designated “ascorbic acid oxidase”. It requires the presence of atmospheric oxygen, which plays the role of hydrogen acceptor. The oxidized ascorbic acid may be reduced to its original state by hydrogen sulphide. The enzyme is remarkably stable to dialysis, oxygen and carbon monoxid. Hydrogen sulphide, however, inactivates it. For ascorbic acid estimation, Tillmans and associates 2 2,6-di-chlorobenzenoneindophenol method was employed.