High and low affinity binding of folate to proteins in serum of pregnancy women.

Binding of [3H]folate to proteins in serum of pregnant women was studied in equilibrium dialysis experiments (pH 7.4, 37 degrees C). A Scatchard analysis revealed the presence of high-affinity (Kass = 10(10) M(-1), N = 0.4 nM folate) and low-affinity sites. The high-affinity folate binding protein (Mr approximately 30 000--35 000) appeared in front effluent after application of serum to a DEAE-Sepharose CL-6B column equilibrated with 0.05 M imidazole buffer (pH 6.3)/30 mM NaCl. Low-affinity binding protein eluted from the column after a rise in NaCl concentration to 1 M was mainly similar to albumin. A minor part was, however, associated with a large molecular size (Mr > 200 000) protein, probably alpha 2-macroglobulin. High-affinity binding which displayed positive cooperativity was saturated at folate concentrations above 10(-10) M. Folate dissociation was a complex process consisting of an initial rapid phase (terminated within 48 h) followed by a slow release. At pH 3.5 dissociation became rapid and complete. Purified methotrexate had no effect on high-affinity binding, whereas N10-methylfolate (an impurity in the methotrexate preparation) acted as a potent inhibitor. Low-affinity binding was proportional to the folate concentration within the range 10(-10)--10(-7) M. Dissociation of folate was rapid.

Change in binding properties of folate-binding protein in cow's whey due to removal of a cofactor during affinity chromatographic purification.

The folate-binding protein in cow's whey was purified by affinity chromatography on folate or methotrexate-AH-Sepharose 4B. A change in basic binding properties of the protein occurred probably due to the fact that material removed during affinity chromatography contains a cofactor of great importance to folate binding.

High-affinity binding of folate to a protein in serum of male subjects.

The binding pattern of [3H]folate in the serum of male subjects was studied in equilibrium dialysis experiments (pH 7.4, 37 degrees C). It was possible to distinguish between a high (association constant, Kass = 10(10) (mol/l)-1; maximum binding of folate = 0.6 nmol/l) and a low affinity type of binding. The high-affinity binder was a trace protein (molecular size approximately 35 000) which was eluted with 30 mmol/l NaC, (pH 6.3) from serum subjected to DEAE-Sepharose CL-6B chromatography. The low-affinity binding activity mainly associated with albumin was eluted with 1 mmol/l NaCl. High-affinity binding was depressed at 7 degrees C (Kass = 10(9) (mol/l)-1). Furthermore, methotrexate acted as a weak inhibitor of high-affinity binding, the molar methotrexate/folate ratio being 100/1 at 50% inhibition.

Pregnancy-specific beta 1-glycoprotein (SP1) determined by means of electroimmunoassay, radial immunodiffusion and nephelometry.

A comparison has been made between rocket-immunoelectrophoresis (RIE), radial immunodiffusion (RID) and automated immunonephelometry (AIP) in the assay of pregnancy-specific beta 1-glycoprotein (SP1) in serum from pregnant women. Using RIE an interaction was demonstrated between the various SP1-reactive molecular populations causing a bias of up to 10%. An interaction corresponding to this phenomenon cannot be demonstrated when using RID and AIP. When correlating the serum-SP1 concentration of samples containing various ratios of SP1-reactive molecules by means of RIE, RID and AIP, it was demonstrated that there was no correlation between the results achieved using one method compared to the results achieved by either of the other methods. The results achieved using one method can therefore exclusively be judged from reference values determined using the same method. The analysis time is essentially shorter with AIP than with RIE and RID.

A high-affinity folate binding protein in normal human leukocytes: ligand binding characteristics, ionic charge and molecular size.

High-affinity binding of [3H]folate to supernatant from homogenized human leukocytes containing large amounts of binding protein displayed apparent positive cooperativity. The DEAE-Sepharose CL-6B chromatographic profile of the supernatant at pH 6.3 contained a major peak of folate binding (Mr approx. 25 000) in the front effluent and a smaller more acidic peak (Mr approx. 25 000) that emerged after a rise in NaCl from 30 mmol/l to 1 mol/l. Triton X-100 solubilized cell sediment from the leukocyte homogenate contained some high-affinity folate binding activity (Mr approx 25 000), typically 5-10% of the total binding activity.

Inheritance of high leukocyte folate binding activity. Relationship between folate binding in leukocytes and serum.

Folate binding was determined in leukocyte lysates from relatives of three persons whose leukocytes contained large amounts of folate binding protein. A pedigree analysis showed that high leukocyte folate binding activities occurred with great frequency in these families suggesting a dominant mode of inheritance. Folate binding in serum varied within narrow limits (0.2-0.5 nmol folate/l serum) in persons with leukocyte folate binding activities less than 0.8-1.0 nmol folate per 10(9) cells. A shift to a higher level of serum folate binding (greater than or equal to 1.0 nmol/l serum) was, however, observed at leukocyte folate binding activities above that value.

Quantitative and qualitative effects of N10-methylfolate on high-affinity folate binding in human leukocytes.

N10-methylfolate acted as a potent competitive inhibitor of high-affinity [3H] folate binding in human leukocytes, while methotrexate had no effect. Furthermore, folate binding changed into a non-cooperative type in the presence of N10-methylfolate. Hence, in qualitative and quantitative respects, the substrate specificity characteristics of leukocyte folate binding resemble those of other high-affinity folate binding systems.

Maximum folate binding in leukocyte lysates and sera from blood donors.

Maximum binding of [3H] folate was determined in leukocyte lysates and sera from blood donors with normal hematological parameters. The folate binding activity in leukocyte lysates showed a remarkably great interindividual variation, whereas the folate binding in sera from the same persons showed a rather modest interindividual variation. The folate binding activity in leukocyte lysates is mainly derived from granulocytes since lymphocytes and monocytes from individuals with high content of folate binder in their leukocytes contained less than 1% of the total binding activity.

Gel filtration profile of folate binding proteins in Triton X-100 solubilized human raw milk.

Solubilization of human raw milk with Triton X-100 gave rise to a two-fold increase in maximum [3H] folate binding as determined in equilibrium dialysis experiments (pH 7.4, 37 degrees C). Gel filtration studies on Triton X-100 solubilized human milk revealed three distinct peaks of protein-bound [3H] folate, Mr approximately 25 000 (38%), Mr greater than or equal to 130 000 (15%) and Mr approximately 100 000 (47%). The first peak was identical to the folate binder previously isolated from human whey, while the second peak most likely represented a particulate membrane-bound form of that binder. The third so far undescribed peak (Mr greater than or equal to 100 000) seemed to contain hydrophobic residues, and might be encapsulated in Triton X-100 micelles. This peak may represent a Triton X-100 solubilized split product of the large particulate binder.

Chromatographic isolation and characterization of folate binding proteins in porcine intestinal epithelial brush border membrane.

Gel filtration studies on solubilized porcine intestinal epithelial brush border membranes labelled with [3H] folate revealed three distinct protein peaks, Mr approximately 25 000, Mr approximately 80 000 and Mr greater than 130 000 (listed in order of decreasing folate binding affinity). The two large molecular size proteins may represent polymerized forms of the Mr approximately 25 000 peak. Folate binding proteins were eluted in front effluent after DEAE-Sepharose CL-6 B chromatography (pH 6.3, 30 mM NaCl) of a solubilized membrane preparation. This means that the cationic forms of these proteins predominate at pH 6.3. In conclusion, folate binding in brush border membranes display characteristics in many respects similar to those of high-affinity folate binding in other tissues and body fluids.

Intended blinders in research.

It could be argued that science is the only generally recognised cognitive authority in the world today. Science is not constrained by frontiers between countries. The art of science is influenced by more factors, how entrenched ideas govern scientific beliefs, also by talent, trends, politics, lobbying, peer-review, priority, conflict of interest etc. Therefore what might have occurred in concealment, is of common scientific interest. An actual example from Denmark is presented showing how research into patients rights and consumer protection has been employed as a technical stumbling-block by health authorities. The authorities problem was certainly a political, not intrinsically a practical medical or scientific one.