A low-cost automated device incorporating a hollow fiber filtration cartridge for large-scale production of ghosts from human erythrocytes.

Readily available elements were used to build an automatic apparatus dedicated to the preparation of erythrocyte ghosts. The apparatus is designed around a low-cost re-usable hollow-fiber filtration cartridge (marketed for therapeutic plasmapheresis). The apparatus is controlled by a simple programmer (based on a diode matrix and low cost timers and liquid level sensors): once the apparatus is loaded with whole red blood cells, washing of cells, as well as hemolysis and washing of ghosts, is performed by the machine in about 4.5 h without any operator intervention. Automatic filter cleaning takes a further 110 min.

New strategies for the screening of a large number of immobilized dyes for the purification of enzymes. Application to the purification of enzymes from human haemolysate.

A method is presented for screening immobilized dyes applicable to the purification of enzymes from haemolysate (haemolysate can be considered as a nearly pure solution of haemoglobin containing only marginal amounts of enzymes). Haemolysate is loaded on immobilized dye mini-columns until haemoglobin and the studied enzymes are found in the column eluate at the same concentrations as those present in the haemolysate. Such a frontal mode of screening allows those dyes to be selected which, displaying a higher affinity for the enzyme of interest than for haemoglobin, can be used to displace the unwanted protein (haemoglobin) from the column by the enzyme of interest (present at a much lower concentration).

Purification of human 6-phosphogluconate dehydrogenase from human haemolysate with chromatography on an immobilized dye as the essential step and use of automation. Simultaneous purification of lactate dehydrogenase.

The screening procedure described in the preceding paper allowed a practical purification procedure to be devised that was automated for human 6-phosphogluconate dehydrogenase. The purification needed only two chromatographic steps, first on immobilized Procion Blue HE-GN and then on Phenyl-Sepharose. This technique also gave purified lactate dehydrogenase. Both enzymes showed single bands in SDS polyacrylamide gel electrophoresis.

Automated purification of human protein band 3, the major integral protein of the erythrocyte membrane.

Human erythrocyte protein band 3 was purified from a Triton X-100 extract of white ghosts. This purification, including an ion-exchange chromatography and a group-affinity chromatography, was automated. The apparatus was assembled from commercially available elements and allowed for the recovery of 2 to 3 mg pure band 3 in 2 hr. The purification could be repeated several times a day. The advantages of automation are discussed.

Protein precipitation induced by a textile dye. Precipitation of human plasminogen in the presence of Procion Red HE3B.

It was demonstrated that human purified plasminogen was precipitated in the presence of the textile dye Procion Red HE3B. The amount of precipitated plasminogen was dependent upon the molar ratio between the dye and protein and seemed to be independent of the protein concentration. A certain amount of dye was coprecipitated with the protein; this was shown also to be related to the dye-to-protein molar ratio. The precipitation of plasminogen induced by the dye was shown to have a pH optimum and to involve ionic and hydrophobic interactions. Procedures were devised which enabled the recovery of precipitated plasminogen in a soluble and non-denatured form totally free from dye. However, the precipitation of plasminogen by Procion Red HE3B could not be used as a single-step purification procedure from a heterogeneous starting material like plasma because of coprecipitation of other proteins. Nevertheless it is suggested that frequently there is a chance that a given protein will be precipitated by a given dye and therefore that protein precipitation by dyes could be a useful complementary method for the purification of proteins.