Isolation of a thermophilic alkaline phosphatase by either hydrophobic or procion red sepharose chromatography

✍ Scribed by Danny Iny; Joseph Sofer; Alex Pinsky

• Publisher: Elsevier Science
• Year: 1986
• Tongue: English
• Weight: 417 KB
• Volume: 360
• Category: Article
• ISSN: 1873-3778
• DOI: 10.1016/s0021-9673(00)91694-7

✦ Synopsis

Afhnity chromatography has become the most efficient method for the purification of active proteins, both with respect to the yield and the extent of enrichmentl. In general, an analogue of the substrate is bonded to an insoluble support such as Sepharose and inactive proteins are eluted under mild solutions, the active factor is eluted under more drastic conditions, usually a higher salt concentration.

When this method was applied to alkaline phosphatase, little success was achieved. Phenylalanine2,3, phosphoric acid3g4 and arsenic acids@ gave poor results. The yield of reagent was poor, and the efficiency of the procedure was low.

Triazine dyes have been used successfully in the purification of many enzymes, especially kinases7s8 and dehydrogenasesg. Their action is similar to that of affinity chromatography. The nucleotide binding fold of the enzyme to be purified apparently reacts with the dye and relatively mild conditions such as NAD, NADH, ATP, phosphate or an increase in salt concentration sutllces to elute the active principle. Thus calf intestinal alkaline phosphatase was eluted from a red Sepharose column with a phosphate gradient of &25 mA4, yielding a final purification of 2500-folds. An organophosphate hydrolysing phosphatase was purified from a Cibacron blue column by a change in the pH of the eluent lo. In the case of the thermophilic alkaline phosphatase described here, elution by much more drastic means such as 4 M guanidine hydrochloride was necessary.

Agarose gels substituted with amino alkanes containing four or more carbon atoms retained phosphorylasel lJ2. Agarose substituted with diamines could be used in the purification of glycogen synthetase 12. From these experiments, it appeared that a protein with hydrophobic pockets will attach itself to a hydrocarbon chain on an insoluble support1 3. Elution is generally achieved by a change in a parameter such as pH or salt concentration13. Thus Nitzan and Michalsky14 succeeded in purifying diphtheria toxin by hydrophobic chromatography.

In the case of the thermophilic alkaline phosphatase described here, none of the above elution methods was successful and as described for the triazine dyes, only 4 A4 guanidine hydrochloride eluted the enzyme. This may be due to the thermophilic nature of the enzyme. Both purification procedures are now described.