Effect of cross-linker length on the stability of hemoglobin.

A series of cross-linking reagents with 4 to 7 carbons have been synthesized and used to modify human hemoglobin. The product yields and biochemical properties of these cross-linked hemoglobins are compared to those made with both longer and shorter cross-linkers. Several trends become apparent. The yields decrease as the cross-linker becomes longer, which correlates well with molecular dynamics studies of reagent binding pathways presented here. The autooxidation rates increase while thermal stability decreases with longer reagents. Cross-linking under deoxy conditions also increases autooxidation rates, but the effect is less than that of increased cross-linker length. The results suggest that shorter reagents may provide better-stabilized tetramers for the construction of more complex hemoglobin-based oxygen carriers.

Introduction of a new regulatory mechanism into human hemoglobin.

Previous studies on bovine hemoglobin (HbBv) have suggested amino acid substitutions, which might introduce into human hemoglobin (HbA) functional characteristics of HbBv, namely a low intrinsic oxygen affinity regulated by Cl(-). Accordingly, we have constructed and characterized a multiple mutant, PB5, [beta(V1M + H2 Delta + T4I + P5A + A76K)] replacing four amino acid residues of HbA with those present at structurally analogous positions in HbBv, plus an additional substitution, beta T4I, which does not occur in either HbBv or HbA. This 'pseudobovine' hemoglobin has oxygen binding properties very similar to those of HbBv: the P(50) of HbA, PB5 and HbBv in the absence of Cl(-) are 1.6, 4.6 and 4.8 torr, respectively, and in 100 mM Cl(-) are 3.7, 10.5 and 12 torr, respectively. Moreover, PB5 has 3-fold slower autoxidation rate compared to HbA and HbBv. These are desirable characteristics for a human hemoglobin to be considered for use as a clinical artificial oxygen carrier. Although the functional properties of PB5 and HbBv are similar, van't Hoff plots indicate that the two hemoglobins interact differently with water, suggesting that factors regulating the R to T equilibrium are not the same in the two proteins. A further indication that PB5 is not a functional mimic of HbBv derives from PB5(control), a human hemoglobin with the same substitutions as PB5, except the beta T4I replacement. PB5(control) has a high oxygen affinity (P(50)=2.3 torr) in the absence of Cl(-), but retains the Cl(-) effect of PB5. The Cl(-) regulation of oxygen affinity in PB5 involves lysine residues at beta 8 and beta 76. PB4, which has the same substitutions as PB5 except beta A76K, and PB6, which has all the substitutions of PB5 plus beta K8Q, both have a low intrinsic oxygen affinity, like HbBv and PB5, but exhibit a decreased sensitivity to Cl(-). Since HbBv has lysine residues at both beta 8 and beta 76, these results imply that Cl(-) regulation in HbBv likewise involves these two residues. The mechanism responsible for the low intrinsic oxygen affinity of HbBv remains unclear. It is suggested that residues peculiar to HbBv at the alpha(1)beta(1) interface may play a role.

Energetic and structural consequences of perturbing Gly-193 in the oxyanion hole of serine proteases.

The oxyanion hole of serine proteases is formed by the backbone N atoms of the catalytic Ser-195 and Gly-193 and engages the backbone O atom of the P1 residue of substrate in an important H-bonding interaction. The energetic contribution of this interaction in the ground and transition states is presently unknown. Measurements of the individual rate constants defining the catalytic mechanism of substrate hydrolysis for wild-type thrombin and trypsin and their G193A and G193P mutants reveal that Gly-193 is required for optimal substrate binding and acylation. Crystal structures of the G193A and G193P mutants of thrombin bound to the active site inhibitor H-d-Phe-Pro-Arg-CH2Cl document the extent of perturbation induced by the replacement of Gly-193. The Ala mutant weakens the H-bonding interaction of the N atom of residue 193, whereas the Pro substitution abrogates it altogether with additional small shifts of the protein backbone. From the kinetic and structural data, we estimate that the H-bonding interaction in the oxyanion hole contributes a stabilization of the ground and transition states of > 1.5 kcal/mol but < 3.0 kcal/mol. These results shed light on a basic aspect of the enzyme-substrate interaction in the entire family of trypsin-like serine proteases.

Thrombin functions through its RGD sequence in a non-canonical conformation.

Previous studies have suggested that thrombin interacts with integrins in endothelial cells through its RGD (Arg-187, Gly-188, Asp-189) sequence. All existing crystal structures of thrombin show that most of this sequence is buried under the 220-loop and therefore interaction via RGD implies either partial unfolding of the enzyme or its proteolytic digestion. Here, we demonstrate that surface-absorbed thrombin promotes attachment and migration of endothelial cells through interaction with alpha(v)beta(3) and alpha(5)beta(1) integrins. Using site-directed mutants of thrombin we prove that this effect is mediated by the RGD sequence and does not require catalytic activity. The effect is abrogated when residues of the RGD sequence are mutated to Ala and is not observed with proteases like trypsin and tissue-type plasminogen activator, unless the RGD sequence is introduced at position 187-189. The potent inhibitor hirudin does not abrogate the effect, suggesting that thrombin functions through its RGD sequence in a non-canonical conformation. A 1.9-Angstroms resolution crystal structure of free thrombin grown in the presence of high salt (400 mm KCl) shows two molecules in the asymmetric unit, one of which assumes an unprecedented conformation with the autolysis loop shifted 20 Angstroms away from its canonical position, the 220-loop entirely disordered, and the RGD sequence exposed to the solvent.

A recombinant polymeric hemoglobin with conformational, functional, and physiological characteristics of an in vivo O2 transporter.

With the objective of developing a recombinant oxygen carrier suitable for therapeutic applications, we have employed an Escherichia coli expression system to synthesize in high-yield hemoglobin (Hb) Minotaur, containing alpha-human and beta-bovine chains. Polymerization of Hb Minotaur through S-S intermolecular cross-linking was obtained by introducing a Cys at position beta9 and substituting the naturally occurring Cys. This homogeneous polymer, Hb Polytaur, has a molecular mass of approximately 500 kDa and was resistant toward reducing agents present in blood. In mice, the circulating half-time (3 h) was fivefold greater than adult human Hb (HbA). The half-time of autooxidation measured in blood (46 h) exceeded the circulating retention time. Hypervolemic exchange transfusion resulted in increased arterial blood pressure similar to that with albumin. The increase in pressure was less than that obtained by transfusion of cross-linked tetrameric Hb known to undergo renovascular extravasation. The nitric oxide reactivity of Hb Polytaur was similar to HbA, suggesting that the diminished pressor response to Hb Polytaur was probably related to diminished extravasation. Transfusion of 3% Hb Polytaur during focal cerebral ischemia reduced infarct volume by 22%. Therefore, site-specific Cys insertion on the Hb surface results in uniform size polymers that do not produce the large pressor response seen with tetrameric Hb. Polymerization maintains physiologically relevant oxygen and heme affinity, stability toward denaturation and oxidation, and effective oxygen delivery as indicated by reduced cerebral ischemic damage.