Recombinant human serum albumin (rHSA) clusters have been synthesized and physicochemically characterized. Cross-linking between the Lys groups of the core albumin and a unique Cys-34 of the shell albumins with an N-succinimidyl-6-[3'-(2-pyridyldithio)propionamido]hexanoate produced the structurally defined rHSA trimer and tetramer. MALDI-TOF-MS showed a single peak with the triple and quadruple masses of rHSA. Their molar ellipticities and the isoelectric points (pI = 4.8) are all identical to those of the monomer, suggesting that the essential structures of the albumin units were intact. TEM observations demonstrated a uniform morphology of the rHSA tetramer with a diameter of 20-30 nm. The circulation half-life (tau1/2) of the 125I-labeled rHSA tetramer in rat (5.5 h) was significantly longer than that of the monomer (2.3 h) due to the low ratio of the distribution phase (alpha-phase). A total of 24 and 32 molecules of the synthetic iron(II) porphyrins (FePs) are incorporated into the hydrophobic cavities of the rHSA trimer and tetramer, respectively, producing huge artificial hemoproteins. These albumin-heme clusters can reversibly bind and release O2 under physiological conditions (37 degrees C, pH 7.3) and showed similar O2-binding properties (O2-binding affinity, association and dissociation rate constants) to those of the corresponding monomer. A large volume of O2 can be chemically dissolved into the albumin-heme cluster solutions relative to the monomeric rHSA-FeP when the molar concentration of the albumin scaffold is identical.