Chromatography of human immunoglobulin G on immobilized drimarene rubine R/K-5BL. Study of mild, efficient elution procedures.

A range of substances were screened to find eluents for human immunoglobulin G (IgG) which are retained with a strong affinity by immobilized Drimarene Rubine R/K-5BL. The strong affinity of IgG for the dye is partly due to the presence of copper in the dye. This was suggested by the effect of substances able to make coordination bonds with metals that elute the IgG and also the effect of metal stripping from the immobilized dye. Several mobile phase conditions were found that allowed desorption of retained IgG on immobilized Drimarene Rubine R/K-5BL without using a protein denaturant. A procedure was also devised for separating IgG2 from other IgG subclasses using chromatography on immobilized Rubine R/K-5BL and column development with an AMP gradient.

Evaluation of several affinity chromatographic supports for the purification of maltose-binding protein from Escherichia coli.

To obtain affinity adsorbents with good mechanical resistance, suitable for the purification of maltose-binding protein (MBP) from Escherichia coli and genetically engineered proteins fused to MBP, a series of supports were prepared by grafting amylose on to agarose by different chemistries. Their capacities for MBP and their abilities to be used at relatively high flow-rates were examined. Efficient supports were most conveniently prepared by coupling amylose to epoxy-activated agarose in an aqueous-organic mixture.

Inhibitory effect of platelet factor 4 (PF4) on the growth of human erythroleukemia cells: proposed mechanism of action of PF4.

The effect of platelet factor 4 (PF4) on the growth of human erythroleukemia cell line (HEL) and the binding characteristics of iodine 125-labeled PF4 to cells were studied to determine the mechanism of action of PF4. HEL cells were cocultured with various doses of PF4 in either a plasma clot system for colony assay or a liquid system for tritiated thymidine incorporation. A significant inhibition of HEL colony growth and tritiated thymidine incorporation was seen at PF4 doses of 1 microgram/ml and 0.5 microgram/ml, respectively. The inhibitory effect of PF4 could be abrogated by the addition of heparin (5 to 10 micrograms/ml). Enzyme-linked immunosorbent assay showed that PF4 had no obvious effect on the expression of platelet glycoprotein IIb/IIIa of HEL cells. Binding of 125I-PF4 to HEL cells reached equilibrium within 20 to 30 minutes with dissociation constant of 1.3 x 10(-10)M and Bmax of 6.3 pmol/10(5) cells and was inhibited by an excess of unlabeled PF4, beta TG, and heparin but was not affected by PMA, IL-3, IL-6, GM-CSF, and interferon-alpha. PF4 did not affect the binding of 125I-IL-3 and 125I-IL-6 to HEL cells. These data demonstrate that PF4 inhibits the growth of HEL cells by specific binding to HEL cells and suggest that the action of PF4 may be associated with the heparin-binding sites of the molecule.

High-performance hydrophobic interaction chromatography of proteins on reversed-phase supports coated with non-ionic surfactants of polyoxyethylene type. Purification of a fungal aspartic proteinase.

On coating reversed-phase supports with polyoxyethylene-type non-ionic surfactants, proteins are no longer retained on such supports at moderate or low ionic strength, but they are retained at high ionic strength and can be desorbed by a decreasing ionic strength gradient. These reversibly modified supports were used for hydrophobic interaction chromatography (HIC). The proteins probably interact with the polyoxyethylene tail of the non-ionic surfactant while the hydrophobic part of the surfactant anchors the surfactant to the reversed-phase support by interactions with its alkane coverage. Although the interactions between non-ionic surfactant and reversed-phase support are non-covalent and the HIC mobile phases contained no surfactant, the modified columns were stable and could be used repeatedly. A surfactant-modified reversed-phase column provided a rapid, efficient, one-step purification of a fungal aspartic proteinase from a commercial crude preparation.

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.

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.

Automated purification of human erythrocytic 6-phosphogluconate dehydrogenase.

Human 6-phosphogluconate dehydrogenase (6PGD) was purified from hemolyzate by group affinity chromatography on 2',5'-ADP-Sepharose, followed by buffer exchange chromatography on Sephadex G-25 and finally salting-out chromatography on Sepharose 6B. An apparatus was assembled from commercially available elements, in which the purification procedure can proceed and be completed unattended and under fully automatic control. The weekly production of the set-up is at present 10 mg of pure 6PGD. The choice of the purification of procedure and the advantages of automation are discussed.