Cholinesterases catalyzing hydrolysis of cholinesters to free choline and the corresponding acids were isolated from bovine erythrocytes and human plasma employing acetylthiocholine as a substrate for the assay of general cholinesterase activity.
The crude preparations of general cholinesterases were then divided into two different enzyme forms based on their substrate specificities, i.e., acetylcholinesterases as a highly specific enzymes for acetylcholine as the substrate, and butyryl(or pseudo)cholinesterase, which has the ability to hydrolyze most cholinesters broadly.
The crude enzyme preparations were treated primarily with ammonium sulfate and D.E.A.E. ion exchange resins, and were purified further by a modern chromatographic technique employing a new affinity column which was prepared by attatching a specific and potent competitive inhibitor of cholinesterases to an insoluble polymer matrix. A ligand molecule of M-dydroxy phenyl trimethyl ammonium iodide was synthesized and attatched to a modified agarose gel derivative $(Affinose 102th)^\textregistered{R}$ for preparation of the useful affinity column.
This approach is based on the biochemical specificity and the unique biological property of proteins to bind with ligand specifically. Thus, the enzyme could be purified easily and effectively to give a reasonably pure form with the highest specific activity.
The final preparations of cholinesterases were obtained as partially pure forms with the specific activities of 4.4 and 6.3 units per mg protein for acetyl cholinesterase and pseudocholinesterase, respectively.
The partially purified enzymes were then immobilized on several organic hydrophilic polymer gels by the entrapping method for the improvement and utilization of the enzyme application. Two different polymer gels of dextrin-starch and polyacrylamide were used as the insoluble polymer backbones.
The condition and technique involved in enzyme immobilization were discussed, and the nature and general physico-chemical properties including the mode of catalytic reaction for both forms of soluble and immobilized cholinesterases were characterized and compared with each other.
A new approach studying enzyme inhibition and its reactivation were also attempted by using the immobilized cholinesterase system. For an another approach to study on pharmacological application, an immobilized acetylcholinesterase on the dextrin-starch gel paper was tested, and it was found to be stable and applicable for the detection of special series of biotoxic derivatives related to the nerve function.
콜린에스테라제는 콜린에스텔을 분해시켜 콜린과 각각의 해당되는산으로 분해시키는 효소로 적혈구와 혈장으로 부터 각각 분리되었다. 전자로부터 얻은 아세틸 콜린에스테라제는 기질로서 주로 아세틸콜린에작용하며 후자에서 얻은 부티릴 콜린에스테라제는 다른 콜린 에스텔에대해서도 친화성을 나타내는 것으로 알려져 있다. 상기의 효소는 일반정제방법과 친화성 크로마토그라피에 의해 정제된 효소로 특히 강한경쟁적 저해제중 하나인 m-hydroxy phenyl trimethyl ammonium iodide 를 ligand 로 하여 프로필렌 디아민이 붙은 Agarose 유도체에 붙인친화성 column 을 이용해 정제 하였다.
이때에 얻은 효소의 비활성도는 아세틸 콜린에스테라제의 경우 4.4 units/mg protein 이였으며 부티릴콜린에스테라제의 경우 6.3units/mg protein 이였다.
위의 정제된 조효소는 친수성 고분자 젤인 덱스트린 전분 젤과 폴리 아크릴라마이드 젤에 각각 불용성화 시켰다. 이들의 고정화된 효소들은 자유형 효소들과 관련하여 물리화학적인 성질의 비교에서 거의 비슷한 것으로 나타났다. 또한 응용면에서는 전자의 지지체인 경우 생체의 신경계와 관련하여 해독물질인 효소저해제의 검출에 사용하였고 후자의 경우 효소의 저해후 재활성화시키는 새로운 해독 연구방법에 이용되어 바람직한 결과를 얻을 수 있었다.
