Preparation of phosphorylcholine derivatives of bovine serum albumin and their application to the affinity chromatography of C-reactive protein.

A simple method for the preparation of phosphorylcholine derivatives of bovine serum albumin (PC-BSA) by reductive alkylation of the amino groups of bovine serum albumin with choline phosphoryl glycoaldehyde is described. Choline phosphoryl glycoaldehyde was generated by periodate oxidation of glyceryl phosphorylcholine. PC-BSA was immobilized on SH-derivatized Toyopearl HW 65 by reacting the single SH group of PC-BSA with a bismaleimido reagent and then coupling maleimidated PC-BSA to the thiolated gel. The affinity purification of C-reactive protein (CRP), which is based on the Ca2+-dependent affinity of CRP for the phosphorylcholine residue of PC-BSA, was readily accomplished using the PC-BSA Toyopearl HW 65 column. The resulting CRP preparation from serum and pleural fluid was homogeneous as assessed by native polyacrylamide gel electrophoresis. PC-BSA derivatives were also shown to be reactive with Limulus polyphemus CRP.

Chromatography in presence of high concentrations of salts on columns of celluloses with and without ion exchange groups (hydrogen bond chromatography). Its application to purification of yeast enzymes.

1. All the water-soluble yeast enzymes tested, which were only partially precipitated at best in the presence of high concentration of salts such as ammonium sulfate or sodium formate, were adsorbed on a column of cellulose in the presence of the same concentrations of the salts, and the adsorbed enzymes were chromatographically eluted by decreasing the concentration of the salts. 2. Even in the presence of high concentration of the salts, the adsorbed enzymes were eluted by urea or by "hydroxy-rich" reagents such as sucrose. 3. Under the experimental conditions used, the salt concentrations required for elution of the adsorbed enzymes were lower with cellulose than with DEAE-cellulose, CM-cellulose, or P-cellulose, indicating that ion exchange groups, either cationic or anionic, affected the adsorption, although the ion exchange groups of DEAE-cellulose, CM-cellulose, and P-cellulose were weakly but definitely functional as ion exchangers even in the presence of high concentrations of the salts. 4. The principal attractive force between cellulose and the enzyme was deduced to be due to hydrogen bonding. 5. This hydrogen bond chromatography was applied for the purification of some yeast enzymes.

Successive purification of several enzymes having affinities for phosphoric groups of substrates by affinity chromatography on P-cellulose.

Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione reductase and pyruvate kinase of Candida utilis and baker's yeast, when in anionic form, were adsorbed on a cation exchanger, P-cellulose, due to affinities similar to those for the phosphoric groups of their respective substrates; thus, glucose-6-phosphate dehydrogenase was readily eluted by either NADP+ or NADPH, glutathione reductase by NADPH, 6-phosphogluconate dehydrogenase by 6-phosphogluconate, and pyruvate kinase by either ATP or ADP. This type of chromatography may be called "affinity-adsorption-elution chromatography"; the main principle is different from that of so-called affinity-elution chromatography. Based on these findings, a large-scale procedure suitable for successive purification of several enzymes having affinities for the phosphoric groups of their substrates was devised. As an example, glucose-6-phosphate dehydrogenase was highly purified from baker's yeast and crystallized.