Phosphate exchange purification

An inactive form of aa-trehalase and its activating protein have been partly purified from resting cells of baker s yeast Saccharomyces) by a combination of fractional precipitation with ammonium sulphate and ion-exchange chromatography. For activation by adenosine 3, 5 -cyclic phosphate, this system appeared to depend on the presence of ATP and Co +, or Mg +, or Mn + cations. The activating protein lost some of its dependence on adenosine 3, 5 -cyclic phosphate during purification, and it may be a protein kinase activation of aa-trehalase would then be associated with phosphorylation of the kinase. 

The purification of the galHum salt solutions is carried out by solvent extraction and/or by ion exchange. The most effective extractants are dialkyl-phosphates in sulfate medium and ethers, ketones (qv), alcohols, and trialkyl-phosphates in chloride medium. Electrorefining, ie, anodic dissolution and simultaneous cathodic deposition, is also used to purify metallic galHum. 

The charged group introduced into products by the aldol donors (phosphate, carboxylate) facilitates product isolation and purification by salt precipitation and ion exchange techniques. Although many aldehydic substrates of interest for organic synthesis have low water solubility, at present only limited data is available on the stability of aldolases in organic cosolvents, thus in individual cases the optimal conditions must be chosen carefully. 

The extraction, purification, or concentration of samples for thaumatin determination will depend on the type of food matrix, as described in Sec. I.C. Purification can also be accomplished by partition (126). Ramsohoye and Kozlov (124) purified thaumatin on a cation-exchange cartridge (CM Sephadex C-50). After conditioning the cartridge with phosphate buffer, the sample is loaded and washed with buffer. Thaumatins are then eluted with 0.25 M sodium chloride. Mackenzie et al. (127) separated thaumatins on cellulose CM52. After equilibration of the column with buffer, pH 7.2, the sample was applied, washed with the buffer, and eluted with a gradient of 0 to 0.2 M NaCl in the same buffer. 

Liquid membranes of the water-in-oil emulsion type have been extensively investigated for their applications in separation and purification procedures [6.38]. They could also allow extraction of toxic species from biological fluids and regeneration of dialysates or ultrafiltrates, as required for artificial kidneys. The substrates would diffuse through the liquid membrane and be trapped in the dispersed aqueous phase of the emulsion. Thus, the selective elimination of phosphate ions in the presence of chloride was achieved using a bis-quaternary ammonium carrier dissolved in the membrane phase of an emulsion whose internal aqueous phase contained calcium chloride leading to phosphate-chloride exchange and internal precipitation of calcium phosphate [6.1]. 

Very recently, Shibayama et al. (1991) reported that cross-linked (a(3)A (chP)aXL isolated by our procedure (i.e., gel filtration in the presence of 1 M MgCl2 followed by ion-exchange chromatography) was found to be contaminated with about 20% of an electrophoret-ically silent impurity that shows higher 02 affinity. They developed a purification technique to separate the desired cross-linked Hb A from the impurity. They found that their purified (aP)A(aP)AXL in the absence of organic phosphate has the same 02 affinity, cooper-ativity, and alkaline Bohr effect as native Hb A. 

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