PubMed · 997712
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J Depersdorff, M Augsten. 1976. [Creatinine determination without deproteinization].. https://pubmed.ncbi.nlm.nih.gov/997712/
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A mechanochemical study of the process of adhesion of plasma proteins to the surface of dialysis membranes was carried out with a scanning force microscope (SFM) in the force spectroscopy mode. Three representative blood plasma proteins (fibronectin, fibrinogen, and albumin) covalently were grafted to a SFM probe, and the adhesion forces of these proteins to cellulosic and synthetic dialysis membranes were measured. The experiment was tailored to apply a controlled load on the protein molecules adsorbed onto the surface in order to simulate the squeezing forces exerted on them during blood filtration. The de-adhesion forces, measured using this new approach for studying the interaction between a protein and dialysis membranes, suggest that the membrane's topography, at a nanometer scale, plays a critical role in the adhesion process. This result was strongly supported by parallel experiments performed on a flattened glass surface with the same dominant hydrophilic character as dialysis membranes. In contrast, a hydrophobic polystyrene surface led to de-adhesion forces at least one order of magnitude greater, overwhelming any possible shape recognition process between the protein molecules and the surface.
Analysis of serum proteins by native polyacrylamide gel electrophoresis is difficult because albumin is abundant in serum and interferes with the resolution of other proteins, especially alpha-antitrypsin which has mobility that is very similar to that of albumin. We present here a method in which serum proteins are separated by polyacrylamide gel electrophoresis using stacking gels containing Blue Sepharose CL-6B, which has a high affinity for albumin, lipoproteins, kinases, and pyridine-nucleotide-dependent oxidoreductases. During electrophoresis, proteins that bind to Blue Sepharose CL-6B stay in the stacking gel and do not migrate into the separating gel. As a consequence, certain proteins, including alpha(1)-antitrypsin, can be detected as clear bands. This method overcomes the requirement for fractionation of serum samples prior to electrophoresis to remove albumin and allows the simultaneous analysis of many samples.
The objectives of this study were to determine the protein binding and lipoprotein distribution of G3139 and G3139 lipoplexes following incubation in human plasma, assess complement activation of, and the effect of pre-incubation of G3139 and G3139 lipoplexes in human plasma on in vitro cellular uptake of G3139. Effect of concentration and time on incorporation of free and lipid associated (lipoplexes) [3H]Bcl-2 AO (25-600 ng/ml) into normolipidemic human plasma lipoproteins was determined by density gradient ultracentrifugation after incubation at 37 degrees C for 5, 30, 60 and 120 min. Protein binding in the lipoprotein deficient fractions (LPDP) was determined by equilibrium dialysis. Complement interaction was determined by ELISA after exposure of human plasma to AO+/- liposomes prepared in serial dilution. In vitro uptake of G3139 and G3139 lipoplexes into human melanoma cells was assessed qualitatively by fluorescence microscopy after 4-h exposure to G3139 (free or as lipoplexes) with or without pre-incubation of G3139 in normal human plasma. Analysis of Bcl-2 AO-lipoprotein interaction over time and concentration indicated no significant movement of the compound within the different lipoprotein and LPDP fractions. Majority of drug was recovered within LPDP fraction, and more than 85% of drug recovered within LPDP fraction was protein bound. No significant activation of complement was noted for either free AO or lipoplexes. Pre-incubation of free AO or AO-lipoplexes in human plasma resulted in a greater cellular uptake of AO-lipoplexes compared with plasma free controls. These findings suggest that the majority of [3H]Bcl-2 AO is plasma protein bound with little lipoprotein association and no significant movement between different lipoprotein and LPDP fractions. Plasma protein binding other than lipoprotein binding may be responsible for the difference in cellular uptake of free AO vs. cationic lipoplexes.