CONSTRUCTION OF BIOLOGICAL PHOSPHORYLATION ANALYZING SYSTEM BASED ON THE CHEMOAFFINITY OF TITANIA

1. (1)高性能二氧化钛颗粒的制备及其理化参数的测定：制备了二氧化钛和二氧化钛等杂交型二氧化钛凝胶。后者对磷酸盐表现出新的保留特性。(2)体外合成和纯化磷酸肽和磷酸蛋白：这些都是酶法制备的。2. 磷酸酯类化合物吸附、解吸、分离和检测条件的研究(1)有机磷在二氧化钛上的吸附和解吸条件：优选条件如下。对于磷酸氨基酸、磷酸肽和核苷酸，使用0.015%三氟乙酸（TFA）吸附在二氧化钛上，然后用含有LiCl（洗脱液A）的50 mM硼酸盐缓冲液（pH 8.0）解吸。对于磷脂，先用氯仿吸附在二氧化钛上，然后用氯仿-异丙醇-磷酸（7:14:5）的混合物解吸。(2)阴离子交换高效液相色谱法分离更多的磷酸化合物的条件：该系统是基于Asahipak NH2P-50柱上的磷酸盐分离，柱后反应涉及磷酸摩尔酸反应。测定了大鼠心肌中有机磷的含量。3. (1)酶解分解磷酸蛋白：在兔磷酸化酶A的胰蛋白酶消化中检测到磷酸肽片段。(2)二氧化钛选择性富集磷酸肽：用0.015% TFA吸附在二氧化钛上，再用A洗脱液解吸，回收率大于80%。(3)阴离子交换高效液相色谱法分离磷酸肽：使用Asahipak nh2o -50色谱柱和A.4洗脱液进行高效分离。构建了以二氧化钛前柱和阴离子交换柱为主体，配备紫外检测器或由紫外检测和OPA柱后荧光检测组成的双检测器系统的磷蛋白分析仪。该系统允许选择性检测和分离微量磷酸肽。少

1. Preparation of materials (1) Preparation of high performance titania particles and determination of their physico-chemical parameters : Titania and some hybrid-type titania gels such as titania-silica were prepated. The latter exhibited novel retention chracteristics for phosphates. (2) in vitro Synthesis and purification of phosphopeptides and phosphoproteins : These were prepared enzymatically. 2. Studies on the conditions for adsorption, desorption, separation and detection of phoshate compounds (1) Conditions for adsorption and desorption of organic phosphates on titania : The Preferable conditions are as follows. For phosphoamino acids, phosphopeptids and nucleotides, adsorbed onto titania using 0.015% trifluoroacetic acid (TFA), and then desorbed by 50 mM borate buffer (pH 8.0) containing LiCl (eluent A). For phospholipids, adsorbed onto titania using chloroform, and then desorbed by a mixture of chloroform-isopropanol-phosphoric acid (7 : 14 : 5). (2) Conditions for separatio … More n of phosphate compounds by anion excahnge HPLC : The system is based on the separation of phosphates on an Asahipak NH2P-50 column and postcolumn reaction involving phosphomolybdic acid reaction. Organic phosphates in rat heart muscle were determind. 3. Development of software for the analysis of phosphoproteins (1) Fragmentation of posphoproteins by enzymatic hydrolysis : A phosphopeptide fragment was detected in tryptic digests of a phosphoprotein, rabbit phosphorylase a. (2) Selective enrichment of phosphopeptides by titania : When adsorbed onto titania using 0.015% TFA and then desorbed by eluent A,the recovery was greater than 80%. (3) Isoration of phosphopeptides by anion exchange HPLC : This is performed efficiently by the use of an Asahipak NH2P-50 column and eluent A.4. Construction of phosphoprotein analyzer Column-switching HPLC systems using a titania precolumn and an anion exchange column, equipped with a UV detector or dual detector system consisted of UV detection and postcolumn fluorescence detection with OPA were developed. The system permitted the slective detection and isolation of trace amounts of phosphopeptides. Less