Purification of pig serum haemopexin by haemin-Sepharose affinity chromatography.

Haemopexin was isolated from pig serum in pure form by affinity chromatography and ion-exchange chromatography. The affinity gel synthesized contained about 0.3 mumol/ml haemin covalently linked to AH-Sepharose 4B. The molecular weight of the protein was measured by polyacrylamide gel electrophoresis in the presence of SDS followed by sensitive silver staining. This procedure and also immunoelectrophoretic studies indicated purity. The mobility of haemopexin in polyacrylamide gels changed following reduction with dithiothreitol.

Spectral and other studies on the intestinal haem receptor of the pig.

We recently demonstrated the presence of a Triton-solubilized high-affinity haem binder on the pig duodenal brush border membrane. The association of haem to the binding factor was determined using radioactive haem and is now studied by a spectrophotometric technique. The binding alters the Soret absorption band of haem from 395 nm to 413 nm. The dissociation constant for the binding of haem to the solubilized binding factor was estimated to be about 10(-9) M by difference spectroscopy. Human serum albumin could not prevent the solubilized binding factor from binding haem. Trypsin digestion destroyed the binder.

Isolation of the haemopexin-haem receptor from pig liver cells.

Isolated pig liver plasma membranes interact specifically with the haemopexin-haem complex (Kd 4.4 X 10(-7) M). Affinity chromatography was used to isolate a membrane component which binds this complex with high affinity. Pig serum haemopexin was first isolated by affinity chromatography on haemin-Sepharose followed by HPLC gel filtration. Liver membranes solubilized with Triton X-100 were incubated with haemin-Sepharose saturated with haemopexin, and as a control, with affinity gel lacking haemopexin. SDS-poly-acrylamide gel electrophoresis of the eluted protein indicated that from the haemin-Sepharose emerglow-molecular-mass haemin-binding proteins whereas the eluate from haemopexin-haemin-Sepharose contained an additional 71 kDa protein, which did not bind free haemin. This protein appears to represent the haemopexin-haem receptor or a part of it. Haem from the haemopexin complex, as also free haemin, was accepted by a binder in the plasma membrane, which in gel filtration behaved like an 80 kDa molecule. This component probably represents a second functional subunit of the haemopexin-haem receptor.

Behaviour in a silica-based high-performance liquid gel permeation chromatographic column of the apo- and holo-forms of the haem-binding proteins haemopexin, histidine-rich glycoprotein, globin and albumin.

The elution of the apo- and holo-forms of four haem-binding proteins was studied using a TSK G 3000 SW HPLC column. Apo-haemopexin had a higher apparent molecular size [68,000 daltons (d)] than histidine-rich glycoprotein (HRG) (66,000 d) in gel chromatography, contrasting with the values in sodium dodecyl sulphate electrophoresis, 84,000 d for HRG and 69,000 d for haemopexin. The elution of the haem complexes of both proteins correlated better with their true molecular weights. Saturation of albumin with haem did not significantly influence its elution. The peaks were more symmetrical for the holo- than the apo-proteins, except for globin/haemoglobin. The results indicated that the apo-forms of haemopexin and HRG had affinity for the column matrix. HRG, which has several haem binding sites, was retained more than haemopexin, which binds only one haem. Free haem itself was bound to the silica column but could be released by globin. HRG had a tendency to polymerize after haem binding, in contrast to haemopexin, which remained monomeric. Globin was eluted from the column with an apparent molecular size of 16,000 d and after saturation with haem with a molecular size of 31,000 d.

Heme-binding plasma membrane proteins of K562 erythroleukemia cells: adsorption to heme-microbeads, isolation with affinity chromatography.

Heme-microbeads attached themselves to the surface of viable K562 cells in a manner inhibitable by free hemin, indicating heme-receptor interaction. The microbeads were at first evenly distributed, but after prolonged incubation at 37 degrees C they formed a cap on one pole of the cells indicating clustering of the membrane heme receptors. Membrane proteins were labeled by culturing the cells in the presence of 35S-methionine and were then solubilized with Triton X-114. The hydrophobic proteins contained about 20% of the total bound label. The solubilized membrane proteins were subsequently adsorbed to a heme-Sepharose affinity gel. According to SDS-electrophoresis and subsequent autoradiography, the immobilized heme captures two proteins or a protein with two polypeptides of 20,000 and 32,000 daltons. The larger of these was only weakly labeled with 35S. The same two bands were observed if the cell surface proteins were labeled with 125I by the lactoperoxidase method and the subsequently solubilized membrane proteins were isolated with heme-Sepharose.

A rosette receptor assay with haem-microbeads. Demonstration of a haem receptor on K562 cells.

A rosette assay was developed for the detection of haem receptor-bearing cells. Indicator particles were prepared by covalent binding of haem to acrylic microbeads. The new method was tested on K562 human erythroleukaemia cells, known to take up haem. In tests on several batches, 80-90% of the K562 cells were rosetted with haem-microbeads whereas mature erythrocytes were haem receptor-negative. Rosette formation was inhibited in a dose-dependent manner by micromolar concentrations of free haemin but not by albumin or transferrin. Uncoated microbeads or albumin-coated microbeads did not attach to K562 cells but transferrin-coated microbeads rosetted 50-70% of them. Diferric transferrin inhibited these rosettes, but haemin had no effect.

Polyclonal activation of human lymphocytes by bacteria.

The proliferative response of human unseparated lymphocytes, T cells, and B cells to various bacterial strains was investigated. All the bacteria tested induced a proliferative response in unseparated lymphocytes and B cells. T cells were stimulated only after reconstitution with monocytes. Stimulation of umbilical cord blood lymphocytes suggests that the response is polyclonal. We interpret these and previous data as an indication of a common mechanism of resistance against infectious diseases.

Studies on the transcobalamin receptor in hog kidney.

The binding of the cobalamin-transcobalamin complex by its solubilized receptor from hog kidney membrane was studied. The receptor bound the complex in a system containing bivalent cations, and the affinity was dependent on the NaCl concentration but not on temperature. The binding of cobalamin-transcobalamin to the receptor had an association constant of approximately 4.6 x 10(9) liter/mol and it was saturable and highly specific as competition by other proteins was not observed. The receptor had higher affinity for the cobalamin-transcobalamin complex (holo-TC) than for transcobalamin (apo-TC). Basic amino compounds known to interfere with tubular reabsorption of proteins did not inhibit the binding. Studies on subcellular fractions supported the view that the receptor was located on the brush border membrane of the kidney.