Solid Ammonium Sulfate Research Articles

Amine Oxidases of Microorganisms

A procedure for obtaining crystalline preparations of the amine oxidase of Aspergillus niger has been developed. The method involved fractionations with ammonium sulfate and separation on successive columns of DEAE-cellulose, DEAE-sephadex and hydroxyl-apatite. Crystals were formed when solid ammonium sulfate was added to solutions of the purified enzyme (of about 300- to 350-times the specific activity of the crude mycelium extract). The crystals appeared as fine rods, with a faint pink color. The crystals had a specific activity around 10,000 which was about 20 times as active as that of crystalline preparations of the animal monoamine oxidase.

L-Lysine Decarboxylase: Preparation of Specific Enzyme and Coenzyme

STRAINS of certain bacteria produce specific amino-acid decarboxylases when grown under acid conditions1. A cell-free preparation of l(+)-lysine decarboxylase has been made from acetone-dried powders of selected strains of coliform organisms grown for 24 hr. at 25° in 2 per cent glucose caseindigest-broth. A suitable paracolon organism, Bact. cadaveris, has been deposited in the National Collection of Type Cultures*. The powder is extracted for 2 hr. at 37° with X ml. M/45 borate buffer pH 8.5 (1 ml. per 20 mgm. powder) and the sediment then centrifuged off. Ethanol is added to the supernatant to a concentration of 20 per cent, the pH. adjusted to 5 with acetic acid and X/2 ml. alumina C? suspension (16 mgm./ml.) added immediately. The alumina is twice eluted with X/2 ml. M/5 phosphate pH. 7.0 and the enzyme precipitated from the combined eluates by the addition of 50 gm. solid ammonium sulphate per 100 ml. The precipitate is dissolved in water and insoluble material centrifuged off. The opalescent supernatant fluid can be used as a source of specific enzyme or purified a further 10–12 times by fractional ammonium sulphate precipitation as shown in the accompanying table.

Attempted Chemical Isolation of the Virus of Poliomyelitis

The demonstration of the chemical nature of the tobacco mosaic virus by Vinson and Petre, its recent isolation as a crystalline protein by Stanley, and the experiments of Howitt indicating that the virus of poliomyelitis may be also a chemical entity, have led us to employ Stanley's procedure in an attempted isolation of the virus of poliomyelitis in the form of a crystalline chemical substance. This attempt was not successful but, because the results are of interest in that they afford added evidence for the theory of the chemical nature of viruses, they are briefly reported herewith. One hundred grams of spinal cords from monkeys infected with the M.V. strain and developing typical paralysis in from 7 to 11 days after intracerebral inoculation, were finely ground with sand and extracted overnight in the refrigerator with 500 cc. of a solution of disodium hydrogen phosphate at pH 8. The thoroughly mixed suspension was poured onto a fine wire screen and the retained precipitate again extracted for 24 hours with 500 cc. of the phosphate solution, poured through the screen and the combined milky suspensions filtered by gravity through coarse fluted filter paper. A monkey inoculated with 0.5 cc. of this filtrate developed typical paralysis in 9 days. The filtrate was weighed and sufficient solid ammonium sulfate added to bring the final concentration to 30% by weight. The precipitate of crude globulin was separated by gravity filtration and extracted twice with 500 cc. portions of phosphate solution at pH 8. The combined filtered extracts were again precipitated with ammonium sulfate at 30% concentration and again extracted with two 500 cc. portions of phosphate solution. The combined filtered extracts were now precipitated with ammonium sulfate at 20% concentration and the globulin dialyzed against distilled water until free from sulfate.

Felton Antibody: its Distribution and Purity as determined by Salting Out Methods

The results obtained show:1. The limits of precipitation of Felton antibody with solid ammonium sulphate in several sera; the effect on these limits of the addition of purified Felton antibody and the removal of tricresol from the serum.2. That within these limits of precipitation small fractions can be obtained which differ from each other in their ratio of antibody to protein.3. That the purity of such fractions throughout a given serum may be reduced by the addition of purified Pneumococcus antibody to the serum, and apparently increased by the addition of tricresol.4. That the distribution of antibody in serum taken at intervals from a horse during immunisation changes progressively.5. Differences in the distribution of the two antibodies in serum from a horse immunised to both Pneumococcus type I and diphtheria toxin.

LIVER FRACTIONS IN PERNICIOUS ANEMIA

Since Minot's1demonstration that a diet high in liver is extraordinarily effective in treating pernicious anemia, we have been endeavoring to find whether certain constituents of liver are as effective as whole liver. We have shown2that a 60 per cent alcoholic extract of liver evaporated to dryness and then washed with absolute alcohol and ether is effective in raising the blood count of patients with pernicious anemia in doses of from 10 to 12 Gm. daily, and that ergothioneine and spermine phosphate are not. The powder derived from the 60 per cent alcohol extraction was dissolved in water and saturated with solid ammonium sulphate. The precipitate proved inactive, but the filtrate, after the excess ammonium sulphate was removed with 60 per cent alcohol, proved active. Doses of 1.6 Gm. daily brought one patient's red cells from 1.4 to 2.7 millions in ten days, the reticulocytes reaching 380,000

THE EXCRETION OF ORGANIC ACIDS AFTER PNEUMONIA

In this paper, it will be shown that large quantities of organic acid are excreted in the urine after the crisis in pneumonia. The patients studied ranged in age from 16 months to 14 years. Eight were typical lobar pneumonia patients; two had bronchopneumonia following influenza. The total quantity of urine in twelve hours was collected, preserved with toluene and kept on ice until analyzed. Analytic methods were: ( a ) determination of total organic acids by the method of van Slyke and Palmer, 1 and ( b ) the determination of ether soluble acids by the following method: Two cubic centimeters of urine was placed in a test tube; 1 gm. of solid ammonium sulphate and 1 drop of concentrated sulphuric acid were added. The ether soluble acids were removed in the continuous ether extractor recently described. 2 Occasionally emulsions formed, and the results of analysis were liable to be too high. No result was