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

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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