COMMUNICATION PATHWAYS IN CENTRAL CAVITY MODIFIED HBS

Project: Research project

Project Details

Description

Many of the synthetic strategies to create hemoglobin based acellular
blood substitutes, employ modifications that perturb the central cavity
and extend the hydrodynamic volume. Cross-bridging (cross-linking),
effector binding and PEGolation (attachment of polyethylene glycols)
result in a wide range of Hbs having a broad spectrum of functional
properties. Some of these properties such as enhanced tetramer stability
and reduced oxygen affinity are needed attributes for potential blood
substitutes. It is our contention that the rational design of this and
the next generation of blood substitutes requires an understanding of how
these modifications couple to functionally relevant domains of the
protein.

Four functionally important intra protein communication pathways are
proposed. Experiments are being pursued to determine how central cavity
modifications and PEGolation affect both key elements of these pathways
and the communication between these elements.

Several optical spectroscopic tools are to be used to probe the static
and dynamic properties of key domains. Visible resonance Raman and near
IR absorption will probe the heme and its immediate environment. Steady
state front face fluorescence, time correlated single photon counting
fluorescence lifetime measurements and nanosecond pulse-probe
fluorescence will be used to probe tryptophans, fluorescent analogues of
DPG and fluorescent probe labeled beta93. UV resonance Raman will be used
both to discriminate between tryptophans and to probe interactions
between the A and E helices the alpha1beta2 interface and the
interactions between the FG corner and the tyrosines of the C terminus.

Iron-metal and meso-proto heme hybrid forms of native, mutant, PEGolated,
cross-bridged and pseudo cross-bridged Hb's will be used to create a
range of tertiary structures and provide a means of probing subunit
specific domains. Subunits with tryptophan replaced with 5-OH tryptophan
will be used to provide subunit specificity for the fluorescence studies.
StatusFinished
Effective start/end date9/30/968/31/02

Funding

  • National Heart, Lung, and Blood Institute
  • National Heart, Lung, and Blood Institute
  • National Heart, Lung, and Blood Institute
  • National Heart, Lung, and Blood Institute: $667,533.00

ASJC

  • Spectroscopy

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