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Features of Boronic Acid μSphere: Ligand: boronic acid Support: polymer beads Spacer: m-aminophenyl group with 9 atome-arm Ligand Density: 75 µMol boronate per ml of settled resin Capacity: at least 99% binding and recovery of 110 µMol AMP per ml of settled resin Format: 50% resin slurry in 50% ethanol. Boronic acid-μSphere is an easy-to-use polymer affinity support for purification of ribonucleotides and other small molecular weight compounds that contain cis-diol groups. The ligand (m-aminophenyl-boronic acid) binds to the cis-diol groups on the sugar portion of nucleotides, forming a reversible five-member ring complex. After washing away non-bound molecules from the sample, the complex can be dissociated and the nucleotide eluted by lowering the pH or by addition of sorbitol. The polymer support used in this product is a macroporous format that allow proteins enter the internal spaces of the beads and bind with the full measure of boronate ligand. Consequently, the resin may purify glycosylated proteins efficiently as it has been used successfully for this application. Boronic acid affinity chromatography has been used to isolate ribonucleoside, to purify nucleosidyl peptide, separate ribonucleosidases in tissue extracts, separate RNA and oligoribonucleotide, and determine of the amount of non-enzymatic glycosylation present in peripheral nerve from diabetic and control rats and dogs. GENERAL REMARKS: Due to the attachment through the amino group, the effective group available to bind proteins is a phenylboronate group, which can form a temporary covalent bond with any molecule that contains a 1,2-cis-diol group. Porath offers a method of preparing the resin by epoxy-activation of beaded agarose. The phenylboronate ligand can be used directly with a molecule containing the cis-diol structure (to produce) a second ligand with more specific binding. Most nucleotides and nucleosides will bind to phenylboronate, so that they, in turn, bind other molecules. These resins can also be used to bind a variety of enzymes, for example, glucose-6-phosphate dehydrogenase and hexokinase, when NADP+ is complexed with the column.3 Lactamases have also been purified using PBA-agarose.4 Serine proteases such as subtilisin, α-chymotrypsin and trypsin have been purified using aminoethyl phenylboronic acid to CH-Sepharose.5 Phenylboronic acid resins have been used for separation and quantitation of glycosylated hemoglobins.6,7 In general, equilibration buffers should be of low ionic strength, with pH 7-9.1 |
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