Paul K Smith

Publications (7)20.52 Total impact

• Article: Hexa-thiocarbamoyl Phenyl PEG5K Hb: Vasoactivity and Structure

  Fantao Meng, Belur N. Manjula, Amy G. Tsai, Pedro Cabrales, Marcos Intaglietta, Paul K. Smith, Muthuchidambaram Prabhakaran, Seetharama A. Acharya
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  ABSTRACT: 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 α-amino groups of Hb, and the other 2 chains are distributed on ε-amino groups, identified as Lys-40(α) (~45%), Lys-56(α) (~25%), and Lys-8(β) (~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 1.9cP and 69.7mmHg, respectively. The molecular radius of (TCP-PEG5K)6-Hb is about 4.6nm 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(β, β′) in (TCP-PEG5K)6-ββ-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.
  The Protein Journal 04/2012; 28(5):199-212. · 1.04 Impact Factor
• Source

  Available from: yu.edu

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

  Fantao Meng, Belur N Manjula, Paul K Smith, Seetharama A Acharya
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  ABSTRACT: 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.
  Bioconjugate Chemistry 08/2008; 19(7):1352-60. · 4.93 Impact Factor
• Source

  Available from: yu.edu

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

  K Ananda, Parimala Nacharaju, Paul K Smith, Seetharama A Acharya, Belur N Manjula
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  ABSTRACT: 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.
  Analytical Biochemistry 04/2008; 374(2):231-42. · 3.00 Impact Factor
• Article: Conjugation of multiple copies of polyethylene glycol to hemoglobin facilitated through thiolation: influence on hemoglobin structure and function.

  Belur N Manjula, Amy G Tsai, Marcos Intaglietta, Ching-Hsuan Tsai, Chien Ho, Paul K Smith, Krishnaveni Perumalsamy, Nirmala Devi Kanika, Joel M Friedman, Seetharama A Acharya
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  ABSTRACT: PEGylation induced changes in molecular volume and solution properties of HbA have been implicated as potential modulators of its vasoconstrictive activity. However, our recent studies with PEGylated Hbs carrying two PEG chains/Hb, have demonstrated that the modulation of the vasoconstrictive activity of Hb is not a direct correlate of the molecular volume and solution properties of the PEGylated Hb and implicated a role for the surface charge and/or the pattern of surface decoration of Hb with PEG. HbA has now been modified by thiolation mediated maleimide chemistry based PEGylation that does not alter its surface charge and conjugates multiple copies of PEG5K chains. This protocol has been optimized to generate a PEGylated Hb, (SP-PEG5K)(6)-Hb, that carries approximately six PEG5K chains/Hb - HexaPEGylated Hb. PEGylation increased the O(2) affinity of Hb and desensitized the molecule for the influence of ionic strength, pH, and allosteric effectors, presumably a consequence of the hydrated PEG-shell generated around the protein. The total PEG mass in (SP-PEG5K)(6)-Hb, its molecular volume, O(2) affinity and solution properties are similar to that of another PEGylated Hb, (SP-PEG20K)(2)-Hb, that carries two PEG20K chains/Hb. However, (SP-PEG5K)(6)-Hb exhibited significantly reduced vasoconstriction mediated response than (SP-PEG20K)(2)-Hb. These results demonstrate that the enhanced molecular size and solution properties achieved through the conjugation of multiple copies of small PEG chains to Hb is more effective in decreasing its vasoconstrictive activity than that achieved through the conjugation of a comparable PEG mass using a small number of large PEG chains.
  The Protein Journal 05/2005; 24(3):133-46. · 1.04 Impact Factor
• Article: Enhanced molecular volume of conservatively pegylated Hb: (SP-PEG5K)6-HbA is non-hypertensive.

  Seetharama A Acharya, Joel M Friedman, Belur N Manjula, Marcos Intaglietta, Amy G Tsai, Robert M Winslow, Ashok Malavalli, Kim Vandegriff, Paul K Smith
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  ABSTRACT: 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.
  Artificial Cells Blood Substitutes and Biotechnology 02/2005; 33(3):239-55. · 0.98 Impact Factor
• Article: WITHDRAWN: PEGylated hemoglobin: Role of surface configuration of PEG for the modulation of hemoglobin vasoactivity.

  Belur N Manjula, Amy G Tsai, Marcos Intaglietta, Ching-Hsuan Tsai, Chien Ho, Ashok Malavalli, Kim D Vandegriff, Robert M Winslow, Paul K Smith, Krishnaveni Perumalsamy, Nirmala Devi Kanika, Joel M Friedman, Seetharama A Acharya
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  ABSTRACT: This paper has been withdrawn since all of the coauthors had not approved of its submission.
  Journal of Biological Chemistry 09/2004; · 4.77 Impact Factor
• Article: Cys-93-ββ-Succinimidophenyl Polyethylene Glycol 2000 Hemoglobin A

  Belur N. Manjula, Ashok Malavalli, Paul K. Smith, Nei-Li Chan, Arthur Arnone, Joel M. Friedman, A. Seetharama Acharya
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  ABSTRACT: Bis(maleidophenyl)-PEG2000 (Bis-Mal-PEG2000), a new bifunctional protein cross-linker targeted to sulfhydryl groups, introduces intra-tetrameric cross-links into oxy-HbA in nearly quantitative yields. Structural as well as crystallographic analyses of the cross-linked species, Bis-Mal-PEG2000 HbA, identified Cys-93(β) as the site of intramolecular cross-linking. The cross-bridging had only a limited influence on the O2 affinity and cooperativity of HbA in 50 mm BisTris acetate, pH 7.4. However, the Bohr effect was reduced by ∼60%. Bis-Mal-PEG2000 HbA retained sensitivity to the presence of allosteric effectors 2,3-diphosphoglycerate, IHP, and chloride, albeit to a lesser degree compared with HbA. Crystallographic analysis revealed the overall structure of deoxy-Bis-Mal-PEG2000 HbA to be similar to deoxy-HbA but for the loss of the salt bridge between Asp-94(β) and His-146(β). The large influence of the cross-bridging on the alkaline Bohr effect of HbA is consistent with the loss of this salt bridge. Unlike the “central cavity cross-bridges” described previously, the cross-link introduced by Bis-Mal-PEG2000 into HbA is an “outside the central cavity cross-bridge.” In view of its oxy-conformational specificity and limited influence on O2 affinity, this new cross-linking strategy holds promise for the stabilization of new designer low O2 affinity Hbs generated by recombinant DNA technology for applications as Hb based therapeutics.
  Journal of Biological Chemistry 02/2000; 275(8):5527-5534. · 4.77 Impact Factor