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We have engineered a recombinant mutant human hemoglobin, Hb Prisca beta(S9C+C93A+C112G), which assembles in a polymeric form. The polymerization is obtained through the formation of intermolecular S-S bonds between cysteine residues introduced at position beta9, on the model of Hb Porto Alegre (beta9Ser --> Cys) (Bonaventura and Riggs, Science 1967;155:800-802). Cbeta93 and Cbeta112 were replaced in order to prevent formation of spurious S&bond;S bonds during the expression, assembly, and polymerization events. Dynamic light scattering measurements indicate that the final polymerization product is mainly formed by 6 to 8 tetrameric hemoglobin molecules. The sample polydispersity Q = 0.07 +/- 0.02, is similar to that of purified human hemoglobin (Q = 0.02 +/- 0.02), consistent with a good degree of homogeneity. In the presence of strong reducing agents, the polymer reverts to its tetrameric form. During the depolymerization process, a direct correlation is observed between the hydrodynamic radius and the light scattering of the system, which, in turn, is proportional to the mass of the protein. We interpret this to indicate that the hemoglobin molecules are tightly packed in the polymer with no empty spaces. The tight packing of the hemoglobin molecules suggests that the polymer has a globular shape and, thus, allows estimation of its radius. An illustration of an arrangement of a finite number of tetrameric hemoglobin molecules is presented. The conformational and functional characteristics of this polymer, such as heme pocket conformation, stability to denaturation, autoxidation rate, oxygen affinity, and cooperativity, remain similar to those of tetrameric human hemoglobin.

The energetic changes that occur on ligand binding in human hemoglobin have been investigated by measurements of the exchange rates of the indole proton of Trpbeta37(C3). The Trpbeta37 residues are located in helices C of the beta-subunits and are involved in contacts with the segments FG of the alpha-subunits at the interdimeric alpha1beta2 and alpha2beta1 interfaces of the hemoglobin tetramer. In the quaternary structure change that accompanies ligand binding to hemoglobin, these contacts undergo minimal changes in relative orientation and in packing, thereby acting as hinges, or flexible joints. The exchange rates of the indole proton of Trpbeta37(C3) were measured by nuclear magnetic resonance spectroscopy, in both deoxygenated and ligated hemoglobin. The results indicate that, at 15 degrees C, the exchange rate is increased from 9.0. 10(-6) to 3.3. 10(-4) s(-1) upon ligand binding to hemoglobin. This change suggests that the structural units at the hinge regions of the alpha1beta2/alpha2beta1 interfaces containing Trpbeta37(C3) are specifically stabilized in unligated hemoglobin, and experience a change in structural free energy of approximately 4 kcal/(mol tetramer) upon ligand binding. Therefore, the hinge regions of the alpha1beta2/alpha2beta1 interfaces could play a role in the transmission of free energy through the hemoglobin molecule during its allosteric transition., (Copyright 2001 Wiley-Liss, Inc.)

The crystal structure of human T state hemoglobin crosslinked with bis(3,5-dibromo-salicyl) sebacate has been determined at 1.9 A resolution. The final crystallographic R factor is 0.168 with root-mean-square deviations (RMSD) from ideal bond distance of 0.018 A. The 10-carbon sebacyl residue found in the beta cleft covalently links the two betaLys82 residues. The sebacyl residue assumes a zigzag conformation with cis amide bonds formed by the NZ atoms of betaLys82's and the sebacyl carbonyl oxygens. The atoms of the crosslink have an occupancy factor of 1.0 with an average temperature factor for all atoms of 34 A2. An RMSD of 0.27 for all CA's of the tetramer is observed when the crosslinked deoxyhemoglobin is compared with deoxyhemoglobin refined by using a similar protocol, 2HHD [Fronticelli et al. J. Biol. Chem. 269: 23965-23969, 1994]. Thus, no significant perturbations in the tertiary or quaternary structure are introduced by the presence of the sebacyl residue. However, the sebacyl residue does displace seven water molecules in the beta cleft and the conformations of the beta1Lys82 and beta2Lys82 are altered because of the crosslinking. The carbonyl oxygen that is part of the amide bond formed with the NZ of beta2Lys82 forms a hydrogen bond with side chain of beta2Asn139 that is in turn hydrogen-bonded to the side chain of beta2Arg104. A comparison of the observed conformation with that modeled [Bucci et al. Biochemistry 35:3418-3425, 1996] shows significant differences. The differences in the structures can be rationalized in terms of compensating changes in the estimated free-energy balance, based on differences in exposed surface areas and the observed shift in the side-chain hydrogen-bonding pattern involving beta2Arg104, beta2Asn139, and the associated sebacyl carbonyl group.

The thermal behavior of the Soret band relative to the carbonmonoxy derivatives of some beta-chain mutant hemoglobins is studied in the temperature range 300-10 K and compared to that of wild-type carbonmonoxy hemoglobin. The band profile at various temperatures is modeled as a Voigt function that accounts for homogeneous broadening and for the coupling with high- and low-frequency vibrational modes, while inhomogeneous broadening is taken into account with a gaussian distribution of purely electronic transition frequencies. The various contributions to the over-all bandwidth are singled out with this analysis and their temperature dependence, in turn, gives information on structural and dynamic properties of the system studied. In the wild-type and mutant hemoglobins, the values of homogeneous bandwidth and of the coupling constants to high-frequency vibrational modes are not modified with respect to natural human hemoglobin, thus indicating that the local electronic and vibrational properties of the heme-CO complex are not altered by the recombinant procedures. On the contrary, differences in the protein dynamic behavior are observed. The most relevant are those relative to the "polar isosteric" beta Val-67(E11)-->Thr substitution, localized in the heme pocket, which results in decreased coupling with low-frequency modes and increased anharmonic motions. Mutations involving residue beta Lys-144(Hc1) at the C-terminal and residue beta Cys-112(G14) at the alpha 1 beta 1 interface have a smaller effect consisting in an increased coupling with low-frequency modes. Mutations at the beta-N-terminal and at the alpha 1 beta 2 interface have no effect on the dynamic properties of the same heme pocket.

A telescopic technique for the end-to-end anastomosis of the common bile duct in the rat is proposed as a way of reducing the number of sutures without prejudicing the impermeability of the union. Serial histological examinations of the scar process after 10, 20, and 30 days showed that patency was maintained.

The brackets in fig 5, show a cluster of cross-linked human and bovine hemoglobins whose oxygen affinity is lower than that of untreated human SFH. Also, they have a stabilized tetrameric structure, and the combination of these characteristics makes these compounds very promising as oxygen carriers in cell-free blood substitutes.