Background: Human serum albumin (HSA) is an abundant plasma protein that binds a wide variety of hydrophobic ligands including fatty acids, bilirubin, thyroxine and hemin. Although HSAheme complexes do not bind oxygen reversibly, it may be possible to develop modified HSA proteins or heme groups that will confer this ability on the complex.

We describe molecular capturing properties of protein nanotubes with a controllable ligand binding affinity and size selectivity. These practical biocylinders were prepared using an alternating layer-by-layer (LbL) assembly of protein and oppositely charged poly(amino acid) into the nanoporous polycarbonate (PC) membrane (pore diameter, 400 nm), with subsequent dissolution of the template. The tube wall typically comprises six layers of poly-L-arginine (PLA) and human serum albumin (HSA) [(PLA/HSA)(3)]. Use of high molecular weight PLA (M(w) = ca. 70 000) yielded robust nanotubes, which are available as lyophilized powder. The (PLA/HSA)(3) nanotubes swelled considerably in water, although the outer diameter was almost unaltered. Uranyl ion, 3,3'-diethylthiacarbocyanine iodide, and zinc(II) protoporphyrin IX (ZnPP) were bound to the HSA component in the cylinder wall. Similar nanotubes comprising recombinant HSA mutant [rHSA(His)], which has a strong binding affinity for ZnPP, captured this ligand more tightly. Furthermore, addition of excess myristic acid released ZnPP from the tubes through a ligand replacement reaction. The hybrid nanotubes bearing a single avidin layer as an internal surface captured FITC-biotin efficiently. Biotin-labeled nanoparticles are also incorporated into the tubes when their particle size is sufficiently small to enter the pores. Subsequent TEM observation revealed a line of loaded nanoparticles (100 nm) in the one-dimensional space interior.

Hemoglobin (Hb, Mw: 64 500) and albumin (Mw: 66 500) are major protein components in our circulatory system. On the basis of bioconjugate chemistry of these proteins, we have synthesized artificial O(2) carriers of two types, which will be useful as transfusion alternatives in clinical situations. Along with sufficient O(2) transporting capability, they show no pathogen, no blood type antigen, biocompatibility, stability, capability for long-term storage, and prompt degradation in vivo. Herein, we present the latest results from our research on these artificial O(2) carriers, Hb-vesicles (HbV) and albumin-hemes. (i) HbV is a cellular type Hb-based O(2) carrier. Phospholipid vesicles (liposomes, 250 nm diameter) encapsulate highly purified and concentrated human Hb (35 g/dL) to mimic the red blood cell (RBC) structure and eliminate side effects of molecular Hb such as vasoconstriction. The particle surface is modified with PEG-conjugated phospholipids, thereby improving blood compatibility and dispersion stability. Manipulation of physicochemical parameters of HbV, such as O(2) binding affinity and suspension rheology, supports the use of HbV for versatile medical applications. (ii) Human serum albumin (HSA) incorporates synthetic Fe(2+)porphyrin (FeP) to yield unique albumin-based O(2) carriers. Changing the chemical structure of incorporated FeP controls O(2) binding parameters. In fact, PEG-modified HSA-FeP showed good blood compatibility and O(2) transport in vivo. Furthermore, the genetically engineered heme pocket in HSA can confer O(2) binding ability to the incorporated natural Fe(2+)protoporphyrin IX (heme). The O(2) binding affinity of the recombinant HSA (rHSA)-heme is adjusted to a similar value to that of RBC through optimization of the amino acid residues around the coordinated O(2).

Covalent core-shell structured protein clusters of hemoglobin (Hb) and human serum albumin (HSA) (HbX-HSAm) (m = 2, 3) with novel physiological properties were generated by linkage of Hb surface lysins to HSA cysteine-34 via an α-succinimidyl-ε-maleimide cross-linker (X: 1 or 2). The isoelectric points of HbX-HSAm (pI = 5.0-5.2) were markedly lower than that of Hb and almost identical to that of HSA. AFM and TEM measurements revealed a triangular Hb1-HSA3 cluster in aqueous medium. The complete 3D structure of Hb1-HSA3 based on TEM data was reconstructed, revealing two possible conformer variants. All HbX-HSAm clusters showed a moderately higher O2 affinity than the native Hb. Furthermore, the exterior HSA units possess a remarkable ability to bind lumiflavin (LF). The addition of NADH to an aqueous solution of the met-Hb2-(HSA-LF)3 cluster reduced the inactive ferric Hb center to the functional ferrous Hb. This O2-carrying hemoprotein cluster with strongly negative surface net charge, high O2 affinity, and NADH-dependent reductase unit can support a new generation of molecular architecture for red blood cell substitutes.

We present the photophysical properties of complexes of recombinant human serum albumin (rHSA) with Zn(II)-protoporphyrin IX (ZnPP) and their activities in the photosensitized reduction of water to hydrogen (H2) using methyl viologen (MV2+) as an electron relay. The ZnPP is bound in subdomain IB of wild-type rHSA [rHSA(wt] by an axial coordination of Tyr-161 and, in the rHSA(I142H/Y161L) mutant [rHSA(His], by a His-142 coordination. Both the rHSA(wt)-ZnPP and rHSA(His)-ZnPP complexes showed a long-lived photoexcited triplet state with lifetimes (tauT) of 11 and 2.5 ms, respectively. The accommodation of ZnPP into the protein matrix efficiently eliminated the collisional triplet self-quenching process. The addition of a water-soluble electron acceptor, MV2+, resulted in a significant decrease in the triplet lifetime. The transition absorption spectrum revealed the oxidative quenching of rHSA-3ZnPP* by MV2+. The quenching rate constant (kq) and backward electron transfer rate constant (kb) were determined to be 1.4 x 10(7) and 4.7 x 10(8) M(-1) s(-1) for rHSA(wt)-ZnPP. In the presence of the colloidal PVA-Pt as a catalyst and triethanolamine (TEOA) as a sacrificial electron donor, the photosensitized reduction of water to H2 takes place. The efficiency of the photoproduction of H2 was greater than that of the system using the well-known organic chromophore, tetrakis(1-methylpyridinium-4-yl)porphinatozinc(II) (ZnTMPyP4+), under the same conditions.

The binding properties of O2 and CO to recombinant human serum albumin (rHSA) mutants with a prosthetic heme group have been physicochemically and kinetically characterized. Iron(III) protoporphyrin IX (hemin) is bound in subdomain IB of wild-type rHSA [rHSA(wt)] with weak axial coordination by Tyr-161. The reduced ferrous rHSA(wt)-heme under an Ar atmosphere exists in an unusual mixture of four- and five-coordinate complexes and is immediately autoxidized by O2. To confer O2 binding capability on this naturally occurring hemoprotein, a proximal histidine was introduced into position Ile-142 or Leu-185 by site-directed mutagenesis. A single mutant (I142H) and three double mutants (I142H/Y161L, I142H/Y161F, and Y161L/L185H) were prepared. Both rHSA(I142H/Y161L)-heme and rHSA(I142H/Y161F)-heme formed ferrous five-N-coordinate high-spin complexes with axial ligation of His-142 under an Ar atmosphere. These artificial hemoproteins bind O2 at room temperature. Mutation at the other side of the porphyrin, Y161L/L185H, also allowed O2 binding to the heme. In contrast, the single mutant rHSA(I142H)-heme could not bind O2, suggesting that removal of Y161 is necessary to confer reversible O2 binding. Laser flash photolysis experiments showed that the kinetics of CO recombination with the rHSA(mutant)-heme were biphasic, whereas O2 rebinding exhibited monophasic kinetics. This could be due to the two different geometries of the axial imidazole coordination arising from the two orientations of the porphyrin plane in the heme pocket. The O2 binding affinities of the rHSA(mutant)-heme were significantly lower than those of hemoglobin and myoglobin, principally due to the high O2 dissociation rates. Changing Leu-161 to Phe-161 at the distal side increased the association rates of both O2 and CO, which resulted in enhanced binding affinity.

A pair of myoglobins containing inherently distorted alpha-ethyl-2,4-dimethyldeuteroheme or undistorted 2,4-dimethyldeuteroheme were prepared, and the functional consequence of intrinsic heme deformation was investigated. The visible absorption peaks of the myoglobin bearing the distorted heme exhibited a bathochromic shift, indicating that the heme was deformed in the protein pocket. Ligand affinities for the ferric myoglobin with the distorted heme were found to be higher than those of the myoglobin bearing the undistorted heme. The observation suggested that the iron atom was more displaced toward the proximal histidine to weaken the coordination of the water molecule. In the paramagnetic proton NMR spectrum of ferrous deoxy protein, the deformed heme caused a 3.2 ppm lower-field shift of the proximal histidine signal, supporting an enhanced iron-histidine interaction. The deformed heme in ferrous myoglobin lowered the oxygen and carbon monoxide affinities by 25- and 480-fold, respectively, and caused the cleavage of the iron-histidine bond in a fractional population of the nitric oxide derivative. These results demonstrate a distinctive controlling mechanism for ligand binding by the deformed heme. Upon the heme distortion, the iron atom is more attracted by the proximal histidine to reduce the affinity of exogenous ligands for the ferrous heme.

A covalent core–shell structured protein cluster composed of hemoglobin (Hb) at the center and human serum albumins (HSA) at the periphery, Hb-HSAm, is an artificial O2 carrier that can function as a red blood cell substitute. Here we described the preparation of a novel Hb-HSA3 cluster with antioxidant activities and its O2 complex stable in aqueous H2O2 solution. We used an approach of incorporating a Pt nanoparticle (PtNP) into the exterior HSA unit of the cluster. A citrate reduced PtNP (1.8 nm diameter) was bound tightly within the cleft of free HSA with a binding constant (K) of 1.1×107 M−1, generating a stable HSA-PtNP complex. This platinated protein showed high catalytic activities for dismutations of superoxide radical anions (O2 •–) and hydrogen peroxide (H2O2), i.e., superoxide dismutase and catalase activities. Also, Hb-HSA3 captured PtNP into the external albumin unit (K = 1.1×107 M−1), yielding an Hb-HSA3(PtNP) cluster. The association of PtNP caused no alteration of the protein surface net charge and O2 binding affinity. The peripheral HSA-PtNP shell prevents oxidation of the core Hb, which enables the formation of an extremely stable O2 complex, even in H2O2 solution.