Enhancing the hydrodynamic volume of Hb

描述（由申请人提供）： PEG化已被证明可降低Hb诱导的血管活性。我们最近开发了一种新的非常有效的聚乙二醇化化学。这种化学反应已用于产生携带六个拷贝的PEG-5000链/四聚体的HbA种类。这种Hb，现在作为HBOC的基于Hb的氧载体以HemostatTM的名称进行测试，具有许多期望的性质，是血管惰性的。实现这些效果所需的最小修饰或甚至需要PEG化HbA的哪些工程性质也不清楚。很明显，PEG化Hb的正性质部分来自其依数性。这个项目旨在了解依数性如何随着PEG化参数的系统调整以及其他尺寸增强策略而变化。 用PEG修饰血红蛋白的表面，使其分子体积/分子质量比显著增加，同时使血红蛋白溶液的粘度和胶体渗透压增加。项目1的总体目标是将由PEG化引起的Hb的分子体积/分子质量比的增加作为PEG链的数量和/或大小、PEG化位点和PEG化化学的函数进行关联，并将PEG化引起的Hb的分子体积/分子质量比的增加与PEG化的分子体积/分子质量比的增加进行交叉关联。 随分子体积/分子质量比变化的依数性Hb的变化。为了实现这一目标，我们致力于设计和评估新的合成策略，以通过三种不同的合成方法产生均匀的尺寸增强的Hbs：1）PEG表面修饰，2）葡聚糖和3）使用基于PEG的双功能试剂的寡聚化。新的单官能PEG试剂类别基于 将合成马来酰亚胺（靶向-SH）、芳基异硫氰酸酯（靶向α-氨基）和酰胺化化学（靶向β和羧基），以实现所需大小PEG链的位点特异性共价连接。还将合成相同PEG试剂的双官能形式用于四聚体间交联。还将开发新的化学物质用于用葡聚糖修饰Hb的表面。结合的潜在优势 将研究新的表面修饰策略，其利用Hb的定点诱变来引入新的反应性官能团（Cys用于反应性Lys）以产生均质产物。这些新的策略将促进蛋白质生物化学核心的努力，以生产材料，将聚乙二醇化血红蛋白的生物化学，依数性和生物物理特性与血管活性的降低相关联，并探索它们之间可能的相互作用。 PEG化介导的Hb性质的改变和Hb的氧和NO反应性的基因工程调节。

DESCRIPTION (provided by applicant): PEGylation has been shown to reduce Hb-induced vasoactivity. We have recently developed a new and very efficient PEGylation chemistry. This chemistry has been used to generate a species of HbA carrying six copies of PEG-5000 strands/tetramer. This Hb, now being tested as an Hb based oxygen carrier of HBOC under the name Hemostat TM, is vaso-inactive with many of desirable properties. It is not clear either as to the minimum modification needed to achieve these effects or even which engineered properties of PEGylated-HbA are required. It is apparent that the positive properties of PEGylated Hb arise in part from their colligative properties. This project seeks to understand how colligative properties vary with systematic tuning of PEGylation parameters as well as other size enhancement strategies. The surface decoration of Hb with PEG increases its molecular volume/molecular mass ration significantly with a concomitant increase in viscosity and colloidal osmotic pressure of the Hb solution. The overall objective of Project 1 is to correlate the increase in the molecular volume/molecular mass ratio of Hb resulting from PEGylation as a function of number and/or size of PEG-chains, site(s) of PEGylation, and PEGylation chemistry and cross-correlate the changes in the colligative properties Hb as a function of changes in the molecular volume/molecular mass ratio. Towards this goal, we undertake to design, and evaluate new synthetic strategies to generate homogeneous size-enhanced Hbs by three different synthetic approaches: surface decoration with 1) PEG, of 2) dextran and 3) oligomerization using PEG-based bifunctional reagents. New classes of monofunctional PEG reagents based on maleimide (targeted to -SH) , aryl isothiocyanate (targeted to a-amino) and amidation chemistry (targeted to beta and carboxyls) will be synthesized to achieve site specific covalent attachment of desired size PEG chains. The bifunctional versions of the same PEG-reagents will also be synthesized for inter-tetrameric cross linking. New chemistries will also be developed for surface decoration of Hb with dextran. The potential advantages of combining new surface decoration strategies with site directed mutagenesis of Hb to introduce new reactive functional groups (Cys for reactive Lys) to generate homogeneous products will be investigated. These new strategies will facilitate the efforts of the Protein Biochemistry Core to produce materials to correlate the biochemical, colligative and biophysical properties of PEGylated Hb with the reduction in vasoactivity, and to explore the possible interplay between the PEGylation mediated changes in the properties of Hb and the genetic engineering modulation of the oxygen and NO reactivity of Hb.