Recombinant Human Serum Albumin Containing 3 Copies of Domain I, Has Significant in Vitro Antioxidative Capacity Compared to the Wild-Type.

Human serum albumin (HSA), the most abundant protein in serum, functions as carrier of drugs and contributes to maintaining serum colloid osmotic pressure. We report herein on the preparation of a genetic recombinant HSA, in which domains II and III were changed to domain I (triple domain I; TDI). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results indicated that the purity of the TDI was equivalent to that of the wild type (WT). Both far- and near-UV circular dichroism (CD) spectra of the TDI showed that its structural characteristics were similar to the WT. Ligand binding capacity was examined by an ultrafiltration method using 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) and ketoprofen as markers for site I and site II, respectively. The binding capacity of TDI for both ligands was lower than that for the wild type. TDI significantly suppressed the oxidation of dihydrorhodamine 123 (DRD) by H2O2 compared to the WT. Our current results suggest that TDI has great potential for further development as HSA a product having antioxidative functions.

S-Nitrosylation of human variant albumin Liprizzi (R410C) confers potent antibacterial and cytoprotective properties.

The S-nitrosylated forms of certain proteins such as albumin have been thought to be circulating endogenous reservoirs of nitric oxide (NO) and may have potential as NO donors in therapeutic applications. In this study, we investigated the characteristics of R410C, a genetic variant of human serum albumin with two free thiols at positions 34 (Cys-34) and 410 (Cys-410), as a NO carrier via S-nitroso formation. A biotin switch assay revealed that Cys-410 was more rapidly and efficiently nitrosylated than was Cys-34. Nitrosylation of Cys-410 introduced only small conformational changes in the protein, which were detected by far-UV circular dichroism but not by near-UV circular dichroism. In addition, both native R410C and S-nitrosylated R410C did not induce molecular heterogeneity through oligomerization. S-Nitrosylated R410C exhibited strong antibacterial activity against Salmonella typhimurium in vitro and suppressed apoptosis of U937 human promonocytic cells induced by Fas ligand. In a rat ischemia-reperfusion liver injury model, S-nitrosylated R410C treatment significantly reduced liver damage, as indicated by markedly decreased release of liver enzymes (aspartate aminotransferase and alanine aminotransferase). Pharmacokinetic analyses indicated retention of the S-nitroso moiety of S-nitrosylated R410C in circulation after i.v. injection, with an approximate half-life of 20.4 min in the mouse. These data suggest that R410C can be a useful NO carrier and can be regarded as a new class of S-nitrosylated proteins possessing antibacterial and cytoprotective properties with a circulation time sufficient for in vivo biological activity.

Recombinant human serum albumin dimer has high blood circulation activity and low vascular permeability in comparison with native human serum albumin.

PURPOSE: Human serum albumin (HSA) is used clinically as an important plasma expander. Albumin infusion is not recommended for critically ill patients with hypovolemia, burns, or hypoalbuminemia because of the increased leakage of albumin into the extravascular spaces, thereby worsening edema. In the present study, we attempted to overcome this problem by producing a recombinant HSA (rHSA) dimer with decreased vascular permeability and an increased half-life. METHODS: Two molecules of rHSA were genetically fused to produce a recombinant albumin dimer molecule. The pharmacokinetics and biodistribution of the recombinant proteins were evaluated in normal rats and carrageenin-induced paw edema mouse model. RESULTS: The conformational properties of this rHSA dimer were similar to those for the native HSA (the HSA monomer), as evidenced by the Western blot and spectroscopic studies. The biological half-life and area under the plasma concentration-time curve of the rHSA dimer were approximately 1.5 times greater than those of the monomer. Dimerization has also caused a significant decrease in the total body clearance and distribution volume at the steady state of the native HSA. rHSA dimer accumulated to a lesser extent in the liver, skin, muscle, and fat, as compared with the native HSA. Up to 96 h, the vascular permeability of the rHSA dimer was less than that of the native HSA in paw edema mouse models. A prolonged plasma half-life of the rHSA dimer was also observed in the edema model rats. CONCLUSIONS: rHSA dimer has a high retention rate in circulating blood and a lower vascular permeability than that of the native HSA.

Functional analysis of recombinant human serum albumin domains for pharmaceutical applications.

PURPOSE: Functional analysis of the three recombinant human serum albumin (rHSA) domains and their potential as stand-alone proteins for use as drug delivery protein carriers. METHODS: Protein structure was examined by fluorescence and CD spectroscopy. Ligand binding was estimated by ultrafiltration. Antioxidant activity was estimated by measuring the quenching of dihydrorhodamine 123. Esterase-like activity and enolase-like activity were estimated from the rate of hydrolysis of p-nitrophenyl acetate and conversion of dihydrotestosterone from the 3-keto to 3-enol form, respectively. The domains of human serum albumin (HSA) were radiolabeled with 111In to evaluate their pharmacokinetics. RESULTS: The ligand binding ability of subsites Ia and Ib could not be detected in domain II. However, the binding of ligands to subsite Ic and site II were preserved in domain II and domain III, respectively. Domain III retained about 45% of its esterase-like activity, and weaker esterase-like activity was also observed in domain I. All domains showed low enolase-like activity in a pH 7.4 phosphate buffer, but domain II had higher activity in a pH 9.2 carbonate buffer. Domain I exhibited antioxidant activity comparable to that of rHSA. All three of the 111In-radiolabeled domains were rapidly eliminated from HSA, with high accumulation in the kidneys. CONCLUSION: Domain I of HSA has great potential for further development as a drug delivery protein carrier, due to its favorable properties and the presence of a free cysteine residue.