The purification of the enzyme hydrolysing diethyl p-nitrophenyl phosphate (paraoxon) in sheep serum.
The purification of the enzyme hydrolysing diethyl p-nitrophenyl phosphate (paraoxon) in sheep serum.
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羊血清中磷酸二乙酯对硝基苯酯（对氧磷）水解酶的纯化。
DOI：
10.1042/bj0740010
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发表时间：
1960
期刊：
影响因子：
--
通讯作者：
A. Main
中科院分区：
文献类型：
--
作者：
A. Main
Aldridge(1953b)报道了哺乳动物血清中的一种酶，它能将乙酰羟乙酯酶抑制剂对硝基苯磷酸二乙酯(对氧磷)水解成二乙基磷酸和对硝基苯酚。以前还没有人认真尝试过从血清或其他组织中提纯这种酶。Aldridge(1953b)用硫酸铵分离兔血清，但没有一个组分有明显的纯化作用。此外，他还证明了科恩等人。(1950)方法10沉淀了(I+II+III)组分中的酶。产率为85%，纯度为2×8倍。Mazur(1946)从兔肾中分离纯化了13倍的水解酶--磷酸二氢丙酯。使用类似的程序，Monter，Floyd和Chanutin(1953)提纯了猪肾二异丙基磷氟化物。ATASE 65-100次。最纯的(A-2)组分由Cohen&Warringa(1957)证明含有两种或可能三种额外的酶。Bergmann，Sgan&Rimon(1957)报道了A-2组分中的一种C-酯酶。根据Aldridge(1953a)的定义，‘C’-酯酶在与A-酯酶基本相同的条件下水解对-硝基苯乙酸酯。Aldridge曾得出结论，血清中对氧磷的水解酶与A-酯酶相同，A-酯酶是对硝基苯乙酸酯的水解酶，不受对氧磷的抑制。然而，由于存在一种在定义A-酯酶的条件下不能水解对氧磷但能水解对硝基苯乙酸酯的酶，这种同一性可能并不在所有情况下都成立。支持这一观点的证据将在后面的一篇关于绵羊血清对氧磷酶的性质的论文中提出，该观点支持不止一种对氧磷酯酶在血清中的水解对硝基苯乙酸酯。对氧磷水解酶被称为对氧磷酶，以区别于其他的A-酯酶。在不纯的组织匀浆中，很难评估这些酶对各种底物的水解的相对贡献。目前的地址：加拿大安大略省渥太华斯宾塞街45号职业健康部门研究小组，使情况变得更加复杂。不同底物和组织的金属离子的影响(Monter&Chanutin，1953,1954)，以及可能存在的自然存在的激活剂和抑制剂(Cohen&Warringa，1957)。部分纯化制剂的使用也引起了不确定性(Cohen&Warringa，1957)。因此，本工作的目的是获得一种均一的制剂，以区分具有相似底物特异性的酶，并在可能的情况下，获得关于该酶的化学性质和可能的生理功能的一些知识。由于对氧磷酶是一种相对不稳定的酶，分离条件温和的纯化程序是可取的。Cohn低温乙醇分馏方法在这方面是令人满意的，但程序没有得到详细遵循。在目前的工作条件下被确定，这简化了Cohn等人。(1946)方法6，同时提高了产率和纯度。此外，希望将血清的稀释度保持在最低限度，以最大限度地利用冷藏设备的容量。在后期阶段，有必要对乙醇、离子强度和pH在低温下的影响进行独立研究。
Aldridge (1953b) reported an enzyme in mammalian sera which hydrolysed the acetyl¢holinesterase inhibitor, diethyl p-nitrophenyl phosphate (paraoxon) to diethylphosphoric acid and p-nitrophenol. No serious attempts have previously been made to purify this enzyme either from serum or from other tissues. Aldridge (1953b) fractionated rabbit serum with ammonium sulphate, but none of the fractions showed significant purification. In addition he showed that the Cohn et al. (1950) method 10 precipitated the enzyme in the (I+ II + III) fraction. The yield was 85% and purification 2*8-fold. An enzyme hydrolysinga related substrate, dii8opropylphosphorofluoridate,waspurified 13-foldfrom rabbit kidney by Mazur (1946). Using a similar procedure, Mounter, Floyd & Chanutin (1953) purified hog-kidney dii8opropyl phosphorofluorid. atase 65-100 times. The most pure (A-2) fraction was shown by Cohen & Warringa (1957) to contain two and possibly three additional enzymes. Bergmann, Segal & Rimon (1957) have reported a 'C'-esterase from the A-2 fraction. 'C'-Esterase hydrolysed p-nitrophenyl acetate under essentially the same conditions as A-esterase, as defined by Aldridge (1953a). Aldridge had concluded that the enzyme hydrolysing paraoxon in serum was identical with A-esterase, which hydrolysed pnitrophenyl acetate and was not inhibited by paraoxon. However, since an enzyme exists which does not hydrolyse paraoxon but does hydrolyse pnitrophenyl acetate under the conditions defining an A-esterase, this identity may not hold in all cases. Evidence supporting the view that more than one paraoxon-resistant esterase hydrolyses pnitrophenyl acetate in serum will be presented in a later paper dealing with the properties of sheepserum paraoxonase. The activity hydrolysing paraoxon has been called paraoxonase to distinguish it from other A-esterases. The relative contributions of these enzymes towards the hydrolysis of various substrates is difficult to assess in impure tissue homogenates. The situation is made more complex by the variable * Present address: Research Group, Occupational Health Division, 45 Spencer Street, Ottawa, Ontario, Canada. effects of metal ions with different substrates and tissues (Mounter & Chanutin, 1953, 1954) and by the possible presence of naturally occurring activators and inhibitors (Cohen & Warringa, 1957). The use of partially purified preparations has also given rise to uncertainties (Cohen & Warringa, 1957). The purpose of the present work was therefore to obtain a homogeneous preparation both to differentiate paraoxonase from enzymes with similar substrate specificities and, if possible, to gain some knowledge about the chemical nature and possible physiological function of this enzyme. Since paraoxonase was a relatively labile enzyme, a purification procedure with mild fractionating conditions was desirable. The Cohn low-temperature ethanol-fractionation approach was satisfactory in this respect, but the procedures were not followed in detail. In the present work conditions were determined which simplified the Cohn et al. (1946) method 6 in the initial stages and at the same time increased both the yield and purification. In addition, it was desired to keep the dilution of the serum to a minimum to utilize the capacity of the refrigerated equipment to best advantage. In the later stages an independent study of the effect of ethanol, ionic strength and pH at low temperatures proved necessary.