Packing density of the PEG-shell in PEG-albumins: PEGylation induced viscosity and COP are inverse correlate of packing density.

PEG-Alb represents a new class of low viscogenic plasma expanders that achieve super perfusion in vivo by mimicking the vasodilatory influence of high viscogenic plasma expanders. PEGylation-engineered structure of PEG albumin can be envisaged as a deformable molecular domain around the rigid central protein core. The correlation between the structure of PEG-shell in terms of packing of the PEG inside the PEG shell and PEGylation induced plasma expander (PE)-like properties of albumin has been investigated as a function of the number and length of the PEG-chain. The increase in molecular radius of albumin on PEGylation is non-linear as a function of the number of PEG chains conjugated. The packing density of PEG within the PEG-shell is an inverse correlate of PEG-chain size; i.e. the shorter chains pack more compactly than the longer ones. The PEGylation induced increase in the viscosity and COP of albumin is an exponential correlation of the number of ethylene oxide units (-CH(2)-CH(2)-O-) conjugated and is also a function of the PEG-chain length. At equivalence of PEG mass conjugated, the viscosity and COP of PEG-albumin adducts correlate inversely with packing density of PEG. All PEGylated albumins are not equivalent on the basis of total PEG mass conjugated. Accordingly, the structure of PEG albumin and its solution properties can be engineered to optimize a given total PEG mass for the application of PEG albumin as a resuscitation fluid. The extension arms minimize the influence of PEG shell on the structure of the protein core. We speculate that EAF-PEGylation is a preferable platform for PEGylation of protein therapeutics and is expected to generate products with better therapeutic efficacy.

Induced plasma expander-like properties as a function of PEG-chains on extension arm facilitated PEGylation of albumin: "mushroom to brush-like" conformational transition of the PEG-albumin conjugate.

Plasma expander-like properties of albumin induced on hexa as well as dodecacPEGylation using Extension Arm Facilitated PEGylation platform make it an excellent resuscitation fluid. PEGylation induced changes in the structure, drug binding, and plasma expander-like properties of bovine serum albumin has been now investigated as a function of PEGylation. The molecular volume of albumin increases on PEGylation nearly linearly; in the beginning up to about six PEG chains are conjugated, then plateau off, while the viscosity and colloidal osmotic pressure change very little initially and then increase exponentially as a function of PEG chains conjugated. PEGylation has essentially no influence on the secondary structure or drug properties of albumin. Tryphtophyl fluorescence of albumin is quenched on PEGylation as a direct correlate of the changes in molecular radius of PEG-albumin. It is concluded that hexaPEGylated and dodecaPEGylated albumin belong to two different configurational states of PEG-albumin in terms of packing of PEG-chains on the molecular surface of the protein. The results suggest a transition of PEGylated albumin from the initial mushroom-like conformation to brush conformation as the PEGylation increases. The therapeutic efficacy of the two PEGylated species is needed to establish the optimum level of PEGylation to function as resuscitation fluids.

Extension arm facilitated pegylation of alphaalpha-hemoglobin with modifications targeted exclusively to amino groups: functional and structural advantages of free Cys-93(beta) in the PEG-Hb adduct.

Cys-93(beta) of hemoglobin (Hb) was reversibly protected as a mixed disulfide with thiopyridine during extension arm facilitated (EAF) PEGylation and its influence on the structural and functional properties of the EAF-PEG-Hb has been investigated. Avoiding PEGylation of Cys-93(beta) in the EAF-PEG-Hb lowers the level of perturbation of heme pocket, alpha1beta2 interface, autoxidation, heme loss, and the O(2) affinity, as compared to the EAF-PEG-Hb with PEGylation of Cys-93(beta).The structural and functional advantages of reversible protection of Cys-93(beta) during EAF PEGylation of oxy-Hb has been compared with Euro PEG-Hb generated by EAF PEGylation of deoxy Hb where Cys-93(beta) is free in the final product. The alphaalpha-fumaryl cross-linking and EAF PEGylation targeted exclusively to Lys residues has been combined together for generation of second-generation EAF-PEG-Hb with lower oxygen affinity. The PEG chains engineered on Lys as well as PEGylation of Cys-93(beta) independently contribute to the stabilization of oxy conformation of Hb and hence increase the oxygen affinity of Hb. However, oxygen affinity of the EAF-PEG-alphaalpha-Hb is more sensitive to the presence of PEGylation on Cys-93(beta) than that of the EAF-PEG-Hb. The present modified EAF PEGylation platform is expected to facilitate the design of novel versions of the EAF-PEG-Hbs that can now integrate the advantages of avoiding PEGylation of Cys-93(beta).

Hexa-thiocarbamoyl phenyl PEG5K Hb: vasoactivity and structure: influence of rigidity of the conjugation linkage on the PEGylation induced plasma expander-like solution properties of PEG-Hb adducts.

A new hexaPEGylated hemoglobin, (TCP-PEG5K)(6)-Hb (TCP, thiocarbamoyl phenyl) has been developed using PEG-phenyl-isothiocyanate and its vasoactivity and structure has been investigated. Of the six PEG5K chains of (TCP-PEG5K)(6)-Hb, 4 are conjugated to the alpha-amino groups of Hb, and the other 2 chains are distributed on epsilon-amino groups, identified as Lys-40(alpha) (approximately 45%), Lys-56(alpha) (approximately 25%), and Lys-8(beta) (approximately 24%). The studies with hamster infused with a bolus of a 4 gm % solution of (TCP-PEG5K)(6)-Hb equivalent to 10% of their blood volume have established that this new hexaPEGylated Hb is vasoinactive. The viscosity and the colloidal osmotic pressure of (TCP-PEG5K)(6)-Hb at 4% is 1.9 cP and 69.7 mmHg, respectively. The molecular radius of (TCP-PEG5K)(6)-Hb is about 4.6 nm and is significantly smaller than hexaPEGylated Hbs developed using other direct and extension arm facilitated PEGylation platform. The presence of an outside the central cavity intramolecular crosslink, succinimidophenyl-PEG2K between Cys-93(beta, beta') in (TCP-PEG5K)(6)-betabeta-Hb strongly impacts its solution properties. These patterns of influence suggest that the inter-dimeric interactions in (TCP-PEG5K)(6)-Hb is weakened just as with other direct PEGylation platforms, and (SP-PEG5K)(6)-Hb generated by EAF-PEGylation is unique in not inducing this effect. A comparison of the properties of hexaPEGylated Hbs establishes that rigidity of the conjugation linkage between PEG and Hb plays a significant influence on the resultant dictating solution properties and/structure/conformation of PEG-Hb adduct.

PEGylation of Val-1(alpha) destabilizes the tetrameric structure of hemoglobin.

A hexaPEGylated hemoglobin (Hb), (Propyl-PEG5K)(6)-Hb, is essentially in alphabeta dimers (Hu et al. (2007) Biochem. J. 402, 143-151). In order to provide a biochemical insight into the tetramer-dimer dissociation of this PEGylated Hb, we prepared and characterized two PEGylated Hbs site-specifically modified at Val-1(alpha) and at Val-1(beta), respectively. PEGylation at Val-1(alpha) and at Val-1(beta) increase the tetramer-dimer dissociation constant (K(d)) of Hb by 2 and 1 order of magnitude, respectively. Accordingly, the sites of PEGylation can determine the tetramer stability of the PEGylated Hb. In order to determine the role of the polyethylene glycol (PEG) chains on the tetramer stability of Hb, we prepared a propylated Hb site-specifically modified at Val-1(alpha). Interestingly, site-specific propylation of Hb at Val-l(alpha) stabilizes the Hb tetramer by 1 order of magnitude. Therefore, conjugation of the PEG chains at Val-1(alpha) can greatly destabilize the tetramer stability of Hb. On the structural aspects, the PEG chains conjugated at Va-1(alpha) unfavorably alter the heme environment and quaternary structure and destabilize the alpha1beta2 interface of Hb. On the functional aspects, the PEG chains conjugated at Val-1(alpha) decrease the Hill coefficient, the Bohr effect of Hb and the sensitization to the presence of the allosteric effectors. In contrast, PEGylation of Hb at Val-1(beta) gives rise to less pronounced structural alteration and different functional change.

Autoxidation of the site-specifically PEGylated hemoglobins: role of the PEG chains and the sites of PEGylation in the autoxidation.

The PEGylated hemoglobin (Hb) has been evaluated as a potential blood substitute. In an attempt to understand the autoxidation of the PEGylated Hb, we have studied the autoxidation of the PEGylated Hb site-specifically modified at Cys-93(beta) or at Val-1(beta). PEGylation of Hb at Cys-93(beta) perturbed the heme environment and increased the autoxidation rate of Hb, which is at a higher level than that caused by PEGylation at Val-1(beta). The perturbation of the heme environment of Hb is attributed to the maleimide modification at Cys-93(beta) and not due to conjugation of the PEG chains. However, the PEG chains enhance the autoxidation and the H 2O 2 mediated oxidation of Hb. Accordingly, the PEG chains are assumed to increase the water molecules in the hydration layer of Hb and enhance the autoxidation by promoting the nucleophilic attack of heme. The autoxidation rate of the PEGylated Hb does not show an inverse correlation with the oxygen affinity. The H 2O 2 mediated structural loss and the heme loss of Hb are increased by maleimide modification at Cys-93(beta) and further decreased by conjugation of the PEG chains. The autoxidation of the PEGylated Hbs is attenuated significantly in the plasma, possibly due to the presence of the antioxidant species in the plasma. This result is consistent with the recent suggestion that there is no direct correlation between the in vitro and in vivo autoxidation of the PEGylated Hb. Therefore, the pattern of PEGylation can be manipulated for the design of the PEGylated Hb with minimal autoxidation.

PEGylation of human serum albumin: reaction of PEG-phenyl-isothiocyanate with protein.

Successful and cost-effective PEGylation protocols require pure functionalized PEG reagents, which can be synthesized by simple and efficient procedures, exhibit high stability against hydrolysis, and maintain a level of reactivity with protein functional groups under mild reaction conditions. PEG-phenyl-isothiocyanate (PIT-PEG) is a new functionalized PEG having these characteristics, and has been synthesized by condensation of the bifunctional reagent 4-isothiocyanato phenyl isocyanate with monomethoxy PEG (mPEG). The data of (1)H NMR and colormetric analysis of the new PEG reagent establish that the mPEG has been quantitatively functionalized. The t 1/4 values for the hydrolysis of PIT-PEG5K in 100 mM phosphate solution at pH 6.5 and 9.2 are about 95 and 40 h, respectively. Incubation of human serum albumin (HSA, 0.5 mM) with a 10-fold molar excess of PIT-PEG (3K or 5K) at pH 6.5 and 9.2 generated PEG-HSA conjugates with average of 3.5 and 6.0 PEG chains per HSA molecule, respectively. The circular dichroism spectra of the conjugates showed that PEGylation of HSA has little influence on the secondary structure of HSA. The hexaPEGylated HSA, (TCP-PEG5K) 6-HSA, exhibited very high hydrodynamic volume, and the molecular radius of HSA increased from 3.95 to 6.57 nm on hexaPEGylation. The hexaPEGylation also increased the viscosity of 4% HSA from 1.05 to 2.10 cP, and the colloid osmotic pressure from 15.2 to 48.0 mmHg. The large increase in the hydrodynamic volume and the solution properties of (TCP-PEG5K) 6-HSA suggest that it could be a potential candidate as a plasma volume expander. PIT-PEG is a useful addition to the spectrum of functionalized PEG reagents available for surface decoration of proteins with PEG.

Non-conservative surface decoration of hemoglobin: influence of neutralization of positive charges at PEGylation sites on molecular and functional properties of PEGylated hemoglobin.

High hydrodynamic volume, high viscosity and high colloidal osmotic pressure (COP) of PEGylated hemoglobin (Hb) have been suggested to neutralize the vasoactivity of acellular Hb. Consequences of non-conservative PEGylation (positive charge of the amino groups at the PEGylation sites is neutralized) using succinimidyl-ester of propionic acid PEG5K on the properties of PEGylated Hb have now been investigated. Non-conservative PEGylation of Hb leads to a much higher increase in the COP and viscosity of Hb than conservative extension arm facilitated (EAF) PEGylation of Hb. Introduction of alphaalpha-fumaryl crosslinking decreased the COP of non-conservative PEGylated Hb by stabilization of interdimeric interactions. Compared to the EAF-PEGylated alphaalpha-fumaryl Hb, non-conservative PEGylated product shows a comparable COP and higher viscosity. Conservative PEGylation of alphaalpha-fumaryl Hb by reductive alkylation chemistry does not increase the COP to this level, but enhanced the molecular volume and viscosity comparable to EAF-PEGylated product. Thus, the molecular properties of PEGylated Hb can be fine tuned using different PEGylation platforms and provide a unique opportunity for the design of second generation PEGylated Hbs.

Analysis of functionalization of methoxy-PEG as maleimide-PEG.

The design of the extension arm-facilitated PEGylation (EAFP) of proteins takes advantage of the high selective and quantitative aspects of the thiol-maleimide reaction. However, the efficiency of EAFP with hemoglobin varied with the batches of maleimide-PEG. The low level of functionalization of monomethoxy-PEG (mPEG) as maleimide-PEG has been now investigated as the potential source of this variation. New chemical approaches for the estimation of the functionalization of mPEG using the reaction of the thiol groups of glutathione, dithiothreitol, and hemoglobin with maleimide-PEG have been developed. The single-step modular approach to the synthesis of maleimidophenyl-PEG (MPPEG) that involved the condensation of p-maleimidophenyl isocyanate with mPEG has been optimized to generate a product with an overall purity of 80%. The NMR approach correlates well with the estimates made by the new chemical approaches. Commercial maleimide-PEG reagents synthesized using multiple steps exhibited a lower level of functionalization as reflected by these chemical estimations. The better functionalization of MPPEG increases the efficiency of EAFP as reflected by the generation of hexaPEGylated Hb and the masking of the D antigen of RBCs. This new EAFP protocol is expected to improve the cost effectiveness of the generation of hexaPEGylated Hb, PEGylated albumin, and PEGylated RBCs as new PEGylated therapeutics.

Non-hypertensive tetraPEGylated canine haemoglobin: correlation between PEGylation, O2 affinity and tissue oxygenation.

TetraPEGylated canine Hb, [SP (succinimidophenyl)-PEG5K]4-canine-Hb, with PEGylation at its four reactive cysteine residues (a111 and b93) has been prepared and characterized. The hydrodynamic volume and the molecular radius of (SP-PEG5K)4-canine-Hb are intermediate to those of di- and hexaPEGylated human Hb as expected. However, the COP (colloidal osmotic pressure) of tetraPEGylated canine Hb is closer to that of hexaPEGylated human Hb than to that of diPEGylated human Hb. The O2 affinity of tetraPEGylated canine Hb is higher than that of canine Hb and comparable with that of hexaPEGylated Hb. The O2 affinity of tetraPEGylated canine Hb is not responsive to the presence of DPG (diphosphoglycerate) or chloride, but it retains almost full response to L-35, an allosteric effector that interacts at the aa-end of the central cavity. The tetraPEGylated canine Hb is vasoinactive in hamster in 10% top load infusion studies. It is also essentially non-hypertensive in an extreme exchange haemodilution protocol in hamster just as di- and hexaPEGylated human Hb. The O2 delivery by tetraPEGylated canine Hb is comparable with that of hexaPEGylated Hb but not as efficient as diPEGylated Hb. These results demonstrate that PEGylation-induced solution properties of PEG [poly(ethylene glycol)]-Hb conjugates are dictated by the level and chemistry of PEGylation and the interplay of these plays a critical role in tissue oxygenation. The studies imply the need to establish the right level (and/or pattern) of PEGylation and O2 affinity of Hb-PEG adducts in designing O2-carrying plasma volume expanders, and this remains the primary challenge in the design of PEGylated Hb as blood substitutes.

Molecular aspects of the high oxygen affinity of non-hypertensive hexa pegylated hemoglobin, [(SP-PEG5K)(6)-Hb].

The development of hexaPEGylated Hb, (SP-PEG5K)(6)-Hb, using the newly designed thiolation-mediated maleimide chemistry based PEGylation, has validated the concept that engineering 'plasma volume expander' -like properties to Hb neutralizes its vasoactivity. The high O(2) affinity of hexaPEGylated Hb has been attributed to the two PEG-5K chains on its two Cys-93(beta) residues. In an attempt to map the influence of the additional four PEG-5K chains of HexaPEGylated Hb on the O(2) affinity, we have now investigated the influence of PEGylation of the surface amino groups alone on the subunit interface interactions and O(2) affinity of Hb using rHb(betaC93A). The molecular radius of PEGylated rHb(betaC93A) was slightly smaller than that of (SP-PEG5K)(6)-Hb, and the overall site-selectivity of PEGylation in the PEGylated rHb(betaC93A) at Lys-residues was comparable to that of (SP-PEG5K)(6)-Hb. Proton NMR studies have shown that the conjugation of the protein with PEG-5K does not have any significant influence on its subunit interface interactions. Surprisingly, the influence of PEGylation on the O(2) affinity and Bohr effect of HbA and rHb(betaC93A) is also nearly the same. Apparently, conjugation of PEG-chains to Lys residues of Hb by the thiolation mediated PEGylation induces unique changes in the structure of the hydration shell of Hb (layer of tightly bound water molecules), which, in turn, induces constraints in its R to T conformational transition to favor the more hydrated R-state.

Thiolation mediated pegylation platform to generate functional universal red blood cells.

The PEGylation that adds an extension arm on protein amino groups with the conservation of their positive charge masks the A and D antigens of erythrocytes efficiently. In the present study, the efficiency of masking the antigens of RBC by PEGylation protocols that do not conserve the charge with and without adding extension arms is compared. The conjugation of PEG-5000 to RBCs through the addition of extension arms masked the D antigen more efficiently than the other protocol. A combination of PEG-5 K and PEG-20 K is needed to mask the A antigen, irrespective of the PEGylation approach. The oxygen affinity of the PEGylated RBCs increased by the extension arm facilitated PEGylation. The protocol involving the conjugation of PEG-chains without adding extension arm did not alter the oxygen affinity of RBCs. A combination of PEGylation protocols is an alternate strategy to generate universal red blood cells with good levels of oxygen affinity.

Combining the influence of two low O2 affinity-inducing chemical modifications of the central cavity of hemoglobin.

HexaPEGylated hemoglobin (Hb), a non-hypertensive Hb, exhibits high O2 affinity, which makes it difficult for it to deliver the desired levels of oxygen to tissues. The PEGylation of very low O2 affinity Hbs is now contemplated as the strategy to generate PEGylated Hbs with intermediate levels of O2 affinity. Toward this goal, a doubly modified Hb with very low O2 affinity has been generated. The amino terminal of the beta-chain of HbA is modified by 2-hydroxy, 3-phospho propylation first to generate a low oxygen affinity Hb, HPPr-HbA. The oxygen affinity of this Hb is insensitive to DPG and IHP. Molecular modeling studies indicated potential interactions between the covalently linked phosphate group and Lys-82 of the trans beta-chain. To further modulate the oxygen affinity of Hb, the alpha alpha-fumaryl cross-bridge has been introduced into HPPr-HbA in the mid central cavity. The doubly modified HbA (alpha alpha-fumaryl-HPPr-HbA) exhibits an O2 affinity lower than that of either of the singly modified Hbs, with a partial additivity of the two modifications. The geminate recombination and the visible resonance Raman spectra of the photoproduct of alpha alpha-fumaryl-HPPr-HbA also reflect a degree of additive influence of each of these modifications. The two modifications induced a synergistic influence on the chemical reactivity of Cys-93(beta). It is suggested that the doubly modified Hb has accessed the low affinity T-state that is non-responsive to effectors. The doubly modified Hb is considered as a potential candidate for generating PEGylated Hbs with an O2 affinity comparable to that of erythrocytes for developing blood substitutes.

Influence of intramolecular cross-links on the molecular, structural and functional properties of PEGylated haemoglobin.

The influence of intramolecular cross-links on the molecular, structural and functional properties of PEGylated {PEG [poly(ethylene glycol)]-conjugated} haemoglobin has been investigated. The sites and the extent of PEGylation of haemoglobin by reductive alkylation are not influenced by the presence of an alphaalpha-fumaryl cross-link at Lys-99(alpha). The propylated hexaPEGylated cross-linked haemoglobin, (propyl-PEG5K)(6)-alphaalpha-Hb, exhibits a larger molecular radius and lower colloidal osmotic pressure than propylated hexaPEGylated non-cross-linked haemoglobin, (propyl-PEG5K)(6)-Hb. Perturbation of the haem microenvironment and the alpha1beta2 interface by PEGylation of haemoglobin is reduced by intramolecular cross-linking. Sedimentation velocity analysis established that PEGylation destabilizes the tetrameric structure of haemoglobin. (Propyl-PEG5K)(6)-Hb and (propyl-PEG5K)(6)-alphaalpha-Hb sediment as stable dimeric and tetrameric molecules, respectively. The betabeta-succinimidophenyl PEG-2000 cross-link at Cys-93(beta) outside the central cavity also influences the molecular properties of haemoglobin, comparable to that by the alphaalpha-fumaryl cross-link within the central cavity. However, the influence of the two cross-links on the oxygen affinity of PEGylated haemoglobin are very distinct, indicating that the high oxygen affinity of PEGylated haemoglobin is not a direct consequence of the dissociation of the haemoglobin tetramers into dimers. alphaalpha-Fumaryl cross-linking is preferred to modulate both oxygen affinity and molecular properties of PEGylated haemoglobin, and cross-linking outside the central cavity could only modulate molecular properties of PEGylated haemoglobin. It is suggested that PEGylation induces a hydrodynamic drag on haemoglobin and this plays a role in the microcirculatory properties of PEGylated haemoglobin.

Pair-wise interactions of polymerization inhibitory contact site mutations of hemoglobin-S.

The linkage of pair-wise interactions of contact site mutations of HbS has been studied using Le Lamentin [His-20 (alpha)-->Gln], Hoshida [Glu-43 (beta)-->Gln] and alpha(2)beta (2) (T87Q) mutations as the prototype of three distinct classes of contact sites of deoxy HbS fiber. Binary mixture experiments established that beta(A)-chain with the Thr-87 (beta)-->Gln mutation is as potent as the gamma-chain of HbF (alpha(2)gamma(2)) in inhibiting polymerization. On combining the influence of Le Lamentin mutation with that of beta (2) (T87Q) mutations; the net influence is only partial additivity. On the other hand, in binary mixture studies, combined influence of Hoshida mutation with that of beta (2) (T87Q) mutations is synergistic. Besides, a significant level of synergistic complementation is also seen when the Le Lamentin and Hoshida mutations are combined in HbS (symmetrical tetramers). Le Lamentin and Hoshida mutation introduced into the cis-dimer of the asymmetric hybrid tetramer completely neutralizes the Val-6 (beta) dependent polymerization. Accordingly, we propose that combining the perturbation of intra-double strand contact site with that of an inter-double strand contact site exhibit synergy when they are present in two different chains of the alphabeta dimer. A comparison of the present results with that of the earlier studies suggest that when the two contact site perturbations are from the same sub-unit of the alphabeta dimer only partial additivity is observed. The map of interaction linkage of the contact site mutations exposes new strategies in the design of novel anti-sickling Hbs for the gene therapy of sickle cell disease.

Dissociation of local nitric oxide concentration and vasoconstriction in the presence of cell-free hemoglobin oxygen carriers.

Cell-free hemoglobin's (CFH) high affinity for nitric oxide (NO) could limit CFH's use as an oxygen-carrying blood replacement fluid because it scavenges NO, causing vasoconstriction and hypertension. However, the extent to which perivascular NO levels change following intravascular administration of hemoglobin (Hb) with different molecular dimensions correlates with vasoconstrictive responses in the microcirculation is unknown. The study objective was to determine vasoconstrictive effects following bolus infusions of (1) alphaalpha cross-linked Hb; (2) polymerized bovine Hb; or (3) polyethylene glycol-decorated Hb (PEG-Hb), by measurements of in vivo microvessel diameter, blood flow, perivascular NO concentration, and systemic hemodynamic parameters. All CFHs caused reductions in perivascular NO levels, not correlated to microvascular responses. PEG-Hb (largest molecular volume) maintained blood flow, while the others caused vasoconstriction and reduced perfusion. All solutions increased mean arterial pressure due to vasoconstriction and blood volume expansion, except for PEG-Hb, which increased blood pressure due to blood volume expansion and maintenance of cardiac output. In conclusion, perivascular NO reduction is similar for all Hb solutions because NO binding affinities are similar; however, effects on vascular resistance are related to the type of molecular modification, molecular volume, and oxygen affinity.

Molecular modeling studies of surface decoration of hemoglobin by maleimide PEG.

Surface decoration of hemoglobin (Hb) with six copies of PEG-5K employing thiolation mediated PEGylation platform neutralizes the vasoaconstricive activity of acellular Hb. The molecular size homogeneity of hexaPEGylated Hb, in spite of the fact that the PEGylation is distributed at multiple sites and PEGylation at each of the sites is not quantitative, is an unusual aspect of this PEGylation reaction. We have introduced three cys residues-Cys-13 (alpha), Cys-111 (alpha), and Cys-13 (beta)-onto Hb by molecular modeling. This new mutant Hb with four reactive Cys residues has been used to build molecular models of PEGylated Hbs with two, four, six, and eight PEG-chains of different masses. The calculated loss of surface area was used to design and gain insight into the structure and the surface shielding of PEGylated Hbs. The modeling shows the adequate surface coverage of the protein hemoglobin with six copies of PEG-5K chains and also exhibits more surface coverage of the hemoglobin as compared to that afforded by two copies of PEG-20K chains.

Extension arm facilitated PEGylation of hemoglobin: correlation of the properties with the extent of PEGylation.

Human hemoglobin (Hb) conjugated with six copies of PEG-5K is nonhypertensive. The hexaPEGylated Hb exhibits molecular size homogeneity in spite of the chemical heterogeneity with respect to the sites of conjugation (Manjula et al., 2005). In the present study, Hb conjugated with an average of 4, 6, 8 and 10 copies of PEG-5K chains have been generated using the extension arm facilitated PEGylation protocol. Except for the tetraPEGylated Hb, all the other products exhibit molecular size homogeneity. The molecular, colligative and functional properties of PEG-Hb conjugates have been correlated with the extent of PEGylation. The results imply that six copies of PEG-5K chains are accommodated on Hb without significant crowding on the molecular surface. As more copies of PEG-5K chains are conjugated to form octa and deca PEGylated Hb, the PEG-chains conjugated appear to undergo transition from a mushroom (compact) to a brush-like conformation (extended conformation) with a concomitant decrease in the propensity of the molecule to transition from oxy to deoxy conformation in the presence of allosteric effectors. The viscosity and the colloidal osmotic pressure of Hb increase with the number of the PEG-chains conjugated in an exponential fashion. The composition of the PEGylated Hb generated appears to be controlled by (i) high reactivity of thiol groups of the extension arms on Hb with maleimide-PEG, (ii) increase in the viscosity of the reaction mixture as the level of PEGylation increases and (iii) increased resistance induced by the PEG-shell of PEGylated Hb to accommodate more PEG-chains as the level of PEGylation increases. Potential implications of extent of PEGylation on the oxygen delivery by PEG-Hb conjugate in vivo have been discussed.

Microvascular effects following treatment with polyethylene glycol-albumin in lipopolysaccharide-induced endotoxemia.

OBJECTIVE: To determine whether resuscitation with polyethylene glycol conjugated bovine serum albumin (2.5% weight/volume) infused at 16 mL/kg/hr (PEG-BSA-16) or at 24 mL/kg/hr (PEG-BSA-24) for 1 hr improves microcirculatory conditions in endotoxemia compared with dextran 70 (6% weight/volume) infused at 24 mL/kg/hr (Dex). DESIGN: Prospective study. SETTING: University research laboratory. SUBJECTS: Male Golden Syrian hamsters. INTERVENTIONS: Hamsters implemented with a skinfold window chamber were given an intravenous injection of lipopolysaccharide and resuscitated within 10 mins with Dex, PEG-BSA-16, or PEG-BSA-24. MEASUREMENTS AND MAIN RESULTS: Hamsters were observed during 24 hrs after lipopolysaccharide injection. Systemic variables measured included mean arterial pressure, heart rate, and systemic arterial blood gas. Microvascular function was characterized by measuring vessel diameter; red blood cell velocity; functional capillary density (FCD); P(O2) in arterioles, venules, and tissue; and perivascular nitric oxide concentration 6 hrs after lipopolysaccharide injection. At 6 hrs, animals with no treatment had the lowest FCD (6.7 +/- 5.7% of baseline). PEG-BSA provided significantly improved microvascular conditions as shown by restoration of FCD. Recovery of FCD was related to improved microvascular flow and perivascular and tissue P(O2), normalization of shear rate, and decreased perivascular nitric oxide concentration. These effects were related to improved fluid retention using PEG-BSA-24 as evidenced by the significantly lower hematocrit at 24 hrs after resuscitation. Nitric oxide at 6 hrs after induction of sepsis achieved perivascular millimolar concentrations, which were reduced to normal values by PEG-BSA-24 treatment. At 6 hrs there were significant differences in FCD, tissue P(O2), and perivascular nitric oxide concentration following PEG-BSA treatment by comparison with Dex treatment, although there were no differences in systemic variables between Dex and PEG-BSA groups. CONCLUSIONS: PEG-BSA produces improved microcirculatory conditions in the treatment of endotoxemia when compared with dextran 70.

Enhanced molecular volume of conservatively pegylated Hb: (SP-PEG5K)6-HbA is non-hypertensive.

Recent studies have suggested that the "pressor effect" of acellular Hb is a consequence of perturbation of the macro-and microcirculatory system in multiple ways, and that PEGylation is an effective approach for controlling the same. In an attempt to confirm this concept, a new and simple thiolation mediated, maleimide chemistry-based conservative PEGylation protocol has been developed to conjugate multiple copies of PEG-chains to Hb. This approach combines the high reactivity of maleimides towards thiols with the propensity of iminothiolane to derivatize the epsilon-amino groups of proteins into reactive thiol groups, with conservation of their positive charge. One of the PEGylated products, namely (SP-PEG5K)6-HbA, that carries on an average six copies of PEG5000 chains per Hb, is non-hypertensive in hamster top load and in rat 50% exchange transfusion models. This hexa-PEGylated-Hb has (i) a hydrodynamic volume corresponding to that of an oligomerized Hb of 256kDa, (ii) a molecular radius of approximately 6.8 nm, (iii) high oxygen affinity, (iv) lowered Bohr effect, and (v) increased viscosity and colloidal osmotic pressure. These properties of (SP-PEG5K)6-HbA are consistent with the emerging new paradigms for the design of Hb based oxygen carriers and confirm the concept that the "pressor effect" of Hb is a multifactorial event. The thiolation mediated maleimide chemistry-based PEGylation protocol described here for the generation of (SP-PEG5K)6-Hb is simple, highly efficient, and is carried out under oxy conditions. The results demonstrate that a non-hypertensive PEG-Hb can be generated by conjugation of a lower number of PEG chains than previously reported.