Evaluating discovery-based research: when biologic reasoning cannot work.

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.

Blood Proteins↗

PubMed2005

Structure of oxidized alpha-haemoglobin bound to AHSP reveals a protective mechanism for haem.

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.

Blood Proteins↗

PubMed2005

Human factor H-related protein 5 has cofactor activity, inhibits C3 convertase activity, binds heparin and C-reactive protein, and associates with lipoprotein.

Factor H-related protein 5 (FHR-5) is a recently discovered member of the factor H (fH)-related protein family. FHR proteins are structurally similar to the complement regulator fH, but their biological functions remain poorly defined. FHR-5 is synthesized in the liver and consists of 9 short consensus repeats (SCRs), which display various degrees of homology to those of fH and the other FHR proteins. FHR-5 colocalizes with complement deposits in vivo and binds C3b in vitro, suggesting a role in complement regulation or localization. The current study examined whether rFHR-5 exhibits properties similar to those of fH, including heparin binding, CRP binding, cofactor activity for the factor I-mediated degradation of C3b and decay acceleration of the C3 convertase. rFHR-5 bound heparin-BSA and heparin-agarose and a defined series of truncations expressed in Pichia pastoris localized the heparin-binding region to within SCRs 5-7. rFHR-5 bound CRP, and this binding was also localized to SCRs 5-7. FHR-5 inhibited alternative pathway C3 convertase activity in a fluid phase assay; however, dissociation of the convertase was not observed in a solid phase assay. rFHR-5 displayed factor I-dependent cofactor activity for C3b cleavage, although it was apparently less effective than fH. In addition, we demonstrate association of FHR-5 with high density lipid lipoprotein complexes in human plasma. These results demonstrate that FHR-5 shares properties of heparin and CRP binding and lipoprotein association with one or more of the other FHRs but is unique among this family of proteins in possessing independent complement-regulatory activity.

Blood Proteins↗