A Micro Manometric Method for the Determination of Diphosphopyridine Nucleotide
نویسنده
چکیده
In the course of histochemical studies of the retina, it was desired to determine the diphosphopyridine nucleotide content of single microtome sections of tissue weighing on the order of 10 to 50 y. It was, therefore, necessary to increase the sensitivity of the manometric method for the determination of DPN. To this end, the technique of Linderstrom-Lang (6) with the Cartesian diver was employed. Of the manometric methods available, the method of Jandorf, Klemperer, and Hastings (4) was chosen for the present purposes owing to the availability and stability of the components of the test system and to the relatively large gas evolution per microgram of DPN. By the micromethod described below 0.001 to 0.006 y of DPN can be determined with an error of less than 5 per cent. This represents a thousandfold increase in sensitivity of the method. Briefly, the principle of the method is as follows: In the presence of arsenate and an extract of acetone powder of cat muscle, the dismutation of triose phosphate to phosphoglyceric acid and phosphoglycerol, normally coupled with the phosphorylation of adenylic acid or adenosine diphosphate, occurs without simultaneous phosphorylation. The dearsenylation of l-arseno-3-phosphoglyceric acid, an intermediate in the over-all reaction, proceeds spontaneously. The molecule of acid formed in this step liberates carbon dioxide from the bicarbonaOe buffer. When DPN is the limiting factor in the system, the rate of gas production is strictly proportional to the concentration of the coenzyme.
منابع مشابه
Automated assay of activities of enzymes involving the diphosphopyridine nucleotide-reduced diphosphopyridine nucleotide reaction.
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