Abstract
In investigating some of the biological effects of oxidases, it was found desirable to prepare active enzymes stable enough to keep for some time. Stephenson, 1 in England, prepared a cell-free extract of B. coli, containing fairly active dehydrogenases, by lysing a concentrated suspension of the organism in hypertonic buffer solution. Bernheim 2 has also reported the preparation of lactic dehydrogenase from acetone yeast. The product, called “Zymin”, gives a fairly active extract and will keep for some time. Gurchot 3 recently described a simple method of extracting lactic dehydrogenase from Prunus seeds. This was published without the knowledge that Thunberg 4 had done the same things previously for other seeds.
We have now found a method for making a stable and potent dry preparation of lactic dehydrogenase, 100 mg. of which will decolorize 0.8 mg. of methylene blue per minute in the presence of lactate. This product was obtained by washing ordinary untreated baker's yeast with saline, and then grinding it with phosphate buffer solution saturated with ether, in a ball mill for 8 to 15 hours. This lysate was centrifuged, cooled in the refrigerator, and shaken with one-half its volume of ether. It was then allowed to stand overnight in the refrigerator. A stable ether gel formed, which had no activity, and which could be separated from the remainder of the solution. This served to remove foreign proteins, and cell debris. After 3 such treatments the liquid was filtered and the solution, which was clear and yellow, was saturated with ammonium sulphate. This was allowed to stand in the refrigerator for one hour and then centrifuged. The precipitate so obtained was dried in a vacuum desiccator.
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