Core--Protein biochemistry

Yeshiva University, New York NY

Investigators

Linked publications & trials

Abstract

DESCRIPTION (provided by applicant): The Protein Biochemistry Core performs the important function of integrating the design strategies of Projects 1 and 2 to produce a blueprint of a matrix of products that can be used for validating the new design paradigms proposed in the Program Project. The success of the individual projects of the Program Project depends heavily on the availability of the various site-specifically surface decorated and inter-tetramerically crosslinked Hbs. This is the responsibility of the Protein Biochemistry Core. The major Core products are 1) Tetrameric Hbs - unmodified HbA mutant Hbs-chemically modified high and low oxygen affinity Hbs, and 2) size-enhancedsHbS namely, the surface decorated Hbs-PEGylated and dextranylated Hbs, and oligomerized Hbs. The native or mutagenized or chemically modified tetrameric Hbs will serve as the starting materials for preparation of the size enhanced Hbs. The Protein Biochemistry Core will receive information from Project 1 on the methodologies for the preparation of the various PEGylated, dextranylated and oligomeric Hbs. A selected set of protocols will be applied to the recombinant Hbs developed in Project 2. Initially, the protocols will be scaled up to generate materials at 1 - 2 g levels. The products generated will be purified and subjected to a variety of biochemical characterization analyses. The materials generated will also be analyzed for their oxygen binding characteristics and supplied to Dr. Vandegriff (Sangart) for characterization of their physical properties- viscosity and colloidal osmotic pressure, and to Projects 2, 3 and 4 for Biophysical investigations. As an initial screen, the materials exhibiting viscosity, COP and hydrodynamic volume comparable to P5K6-HbA will be supplied to Project 5 for screening of their vasoactivity by the top load assay in hamster. The integration of the results of these studies with those from Projects 2, 3 and 4 will lead to the identification of a set of materials for more detailed investigations in the microcirculatory assay (exchange transfusion and hemorrhagic shock models), and for measurements of diffusive oxygen transport in artificial capillaries. Such materials will be generated at 5 - 10 g levels. The Protein Biochemistry Core will coordinate with the BioAffinity Systems for the large-scale synthesis of the PEG reagents. The Core will also provide support for Projects 3 and 4 in the synthesis of specialty -probe labeled surface decorated Hbs for studies on the local and global effects of the surface decoration on the structure of Hb.