PubMed · 3927702
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R M Russell. 1985. Relative dose response test.. https://doi.org/10.1093/ajcn%2F42.2.342
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In the present investigation, the binding of roscovitine (100, 500 and 1500 ng/mL) to plasma proteins was studied at 25 and 37 degrees C by ultrafiltration and equilibrium dialysis methods. Drug stability in plasma was assessed during a 48 h at 4, 25 and 37 degrees C. The effect of thawing and freezing on drug stability was studied. The pKa of roscovitine was measured using capillary electrophoresis coupled with mass spectrometry. Roscovitine was quantified utilizing liquid chromatography and tandem mass spectrometry. Roscovitine is highly bound to plasma proteins (90%). Binding of roscovitine to human serum albumin was constant (about 90%) within concentration range studied while the binding to alpha1-acid glycoprotein decreased with increasing drug concentration indicating that albumin is more important in clinical settings. However, alpha1-acid glycoprotein might be important when plasma proteins change with disease. Protein binding was higher at 25 degrees C compared to 37 degrees C. The results obtained by equilibrium dialysis were in good agreement with those obtained by ultrafiltration. Roscovitine was stable at all temperatures studied during 48 h. Roscovitine has a pKa of 4.4 showing that the drug mainly acts like a weak mono-base. The results obtained in our studies are important prior to clinical trials and to perform pharmacokinetic studies.
Frequent in vivo sampling of blood proteins is often stressful, making it difficult to obtain more than a few samples. As a result, only limited time-profiles can be made. We have developed an ultrafiltration collection device (UCD) for continuous sampling. The UCD consists of a hollow fiber, a coil and a flow creator. Hollow fiber membranes are often hydrophobic and this can result in adsorption of protein and/or peptides, leading to clogged membranes. Adsorption was tested with a hydrophobic and hydrophilic peptide and two biocompatible hollow fibers made from different materials. The hollow fiber made from poly(ethylene) coated with ethylenevinyl alcohol gave near 100% recovery for both peptides. This was in contrast to the poly(sulfone) hollow fiber when sampling the hydrophobic peptide. Filling the coil with various peptide concentrations gave good recovery and insignificant diffusion even after storage for 6 d at 37 degrees C. Continuous pulse-free sampling was tested by vacuum. An average flow rate of 423 +/- 50 nl min(-1) over a period of 4 d was created using S-Monovette. The flow rate gradually declined during this period by <5% every consecutive day. In addition, we also examined a complex sample-serum in the poly(ethylene) hollow fiber. Serum and ultrafiltrate were spotted onto a protein chip and analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Six proteins out of 64 were found to be significantly different between serum and the ultrafiltrate (p < 0.05). The UCD has the potential to be used for in vivo real-time monitoring.
The synthesis of haemoglobin A (HbA) is exquisitely coordinated during erythrocyte development to prevent damaging effects from individual alpha- and beta-subunits. The alpha-haemoglobin-stabilizing protein (AHSP) binds alpha-haemoglobin (alphaHb), inhibits the ability of alphaHb to generate reactive oxygen species and prevents its precipitation on exposure to oxidant stress. The structure of AHSP bound to ferrous alphaHb is thought to represent a transitional complex through which alphaHb is converted to a non-reactive, hexacoordinate ferric form. Here we report the crystal structure of this ferric alphaHb-AHSP complex at 2.4 A resolution. Our findings reveal a striking bis-histidyl configuration in which both the proximal and the distal histidines coordinate the haem iron atom. To attain this unusual conformation, segments of alphaHb undergo drastic structural rearrangements, including the repositioning of several alpha-helices. Moreover, conversion to the ferric bis-histidine configuration strongly and specifically inhibits redox chemistry catalysis and haem loss from alphaHb. The observed structural changes, which impair the chemical reactivity of haem iron, explain how AHSP stabilizes alphaHb and prevents its damaging effects in cells.