The purpose of this study was to dissociate effects of reduced viscosity from those of low arterial O2 content (CaO2) on cerebral blood flow (CBF) during anemia. Three groups (n = 8) of pentobarbital sodium-anesthetized cats were studied: 1) a time-control group with a hematocrit of 32 +/- 1% (SE), 2) an anemia group that underwent an isovolumic exchange transfusion with albumin in a salt solution to decrease hematocrit to 18 +/- 1%, and 3) a group transfused with cell-free, tetramerically stabilized hemoglobin to decrease hematocrit equivalently to that in the albumin-transfused group. CaO2 (in ml/dl) in the hemoglobin-transfused group (11.8 +/- 0.3) and the control group (15.0 +/- 0.6) was greater than that in the albumin group (8.7 +/- 0.3). CBF (in ml.min-1.100 g-1) in the hemoglobin group (45 +/- 3) and control group (36 +/- 4) was less than that in the albumin group (60 +/- 3). Consequently, cerebral O2 transport (CaO2 x CBF) was similar in the hemoglobin, control, and albumin groups (5.3 +/- 0.3, 5.3 +/- 0.4, and 5.2 +/- 0.2 ml.min-1.100 g-1, respectively). After infusion of N omega-nitro-L-arginine methyl ester (L-NAME) to inhibit nitric oxide (NO) synthase, CBF in the hemoglobin group remained lower than that in the albumin group, suggesting that NO scavenging by hemoglobin did not solely account for the lower CBF. In contrast, the neurohypophysis (posterior pituitary) exhibited substantial decreases in blood flow that were not augmented by L-NAME administration after hemoglobin transfusion and that were similar in magnitude to L-NAME alone. Thus NO scavenging by cell-free hemoglobin may be more prominent in high-flow, protein-permeable regions enriched with NO synthase. These results support the hypothesis that O2 transport to cerebrum is well regulated when CaO2 is manipulated independently of hematocrit and viscosity.

Cross-linked human hemoglobin (HbA) is obtained by reaction with bis(3,5-dibromosalicyl) sebacate. Peptide maps and crystallographic analyses confirm the presence of the 10 carbon atom long sebacyl residue cross-linking the two beta82 lysines of the beta-cleft (DecHb). The Adair's constants, obtained from the oxygen binding isotherms, show that at the first step of oxygenation normal hemoglobin and DecHb have a very similar oxygen affinity. In DecHb negative binding cooperativity is present at the second step of oxygenation, which has an affinity 27 times lower than at the first step. Positive cooperativity is present at the third binding step, whose affinity is 380 times that of the second step. The fourth binding step shows a weak negative cooperativity with an affinity one-half that of the third step. Crystals of deoxy-DecHb diffracted to 1.9 angstroms resolution. The resulting atomic coordinates are very similar to those of Fermi et al. [(1984) J. Mol.Biol. 175, 159-174] and Fronticelli et al. [(1994) J. Biol Chem. 269, 23965-23969] for deoxy-HbA. The electron density map of deoxy-DecHb indicates the presence of the 10 carbon bridge between the beta82 lysines. Molecular modeling confirms that insertion of the linker into the T structure requires only slight displacement of the two beta82 lysines. Instead, insertion of the linker into the R and R2 structures [Shaanan (1983) J. Mol. Biol. 171, 31-59; Silva et al. (1992) J. Biol. Chem. 267, 17248-17256] is hindered by serious sterical restrictions. The linker primarily affects the partially and fully liganded states of hemoglobin. The data suggest in DecHb concerted conformational changes at each step of oxygenation.

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