Formation of L-Glutamic Acid by the α-Ketoglutaric Leucine Transaminase of Saprogenic Bacteria (IV) : An Optical Method for the Simultaneous Estimation of α-Ketoglutaric Acid (α-KG) and α-Ketoisocaproic Acid (α-KIC)

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  • 腐敗細菌のα-ケトグルタール酸-ロイシン系トランスアミナーゼに依るL-グルタミン酸の生成に就いて (第4報) : α-ケトグルタール酸-α-ケトイソカプロン酸の分別定量法について

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The simultaneous estimation of α-KG and α-KIC was studied for demonstrating the α-KG-leucine transamination reaciton. For the estimation of two acids, the modified method of FRIEDEMANN and HAUGEN was applied. The reaction rate of α-KIC with 2,4-dinitrophenylhydrazine (DiNP) was larger than that of α-KG as well as pyruvic acid. Moreover, it was shown that α-KIC-2,4-dinitrophenylhydrazone (α-KIC-DiNPH) was not extractable with 10% Na_2CO_3 solution from ethyl acetate solution.Based on these facts, several conditions for the estimation of the two α-keto acids were studied. First, α-KIC was subject to the reaction with DiNP, for 15 minutes at 5℃ and α-KIC-DiNPH formed was extracted from the mixture with 3ml of toluene under shaking for 3 minutes. The residue was added again with DiNP and incubated for 10 minutes at 40℃, and α-KG-DiNPH was extracted with 8ml of ethyl acetate for 4 minutes.Then, the two DiNPH derivatives of α-Keto acids in the toluene layer and ethyl acetate layer were transferred to 6ml of 10% Na_2CO_3 solution. To the 5ml of 10% Na_2CO_3 layer, 2ml of 4N NaOH was added and the optical density was estimated at 517mμ in a spectrophotometer within 2～3 minutes after color development.As the separation of α-keto acids by the above method was not complete, the estimated values should be corrected in order to minimize errors. An equation for such a correction was proposed.

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