This proposal is a continuation of our research in understanding the adsorption behavior of proteins and other biopolymers on chromatographic surfaces. Three phases of research are proposed. (1) First, we shall continue our work on the study of the behavior of proteins on surfaces using intrinsic fluorescence spectroscopy. We have already been able to determine the kinetic mechanism of unfolding of alpha-lactalbumin on weakly hydrophobic surfaces. We proposed to examine with this species and other globular proteins the role of various parameters on the thermodynamics of interaction with tailor-made surfaces and the kinetics of the unfolding processes. These parameters will include mobile phase additives such as metal ions, structure of the adsorbent surface, protein surface concentration and nonporous vs. porous beads. (2) We propose to purchase a scanning lifetime fluorescence spectrometer in order to probe more deeply protein conformational changes on adsorbent surfaces. Through lifetime measurements and the dynamics of anistropic decay, we will explore the environment around individual fluorophores as a function of contact time with the surface. Such measurements will allow probing different portions of the protein as structural change occurs. (3) In collaboration with Genentech, we propose to explore the chromatographic and intrinsic fluorescence surface behavior of recombinant growth hormone and a large number of variants. Both point mutations and mutations of sequences of amino acids will be examined. These studies will aid in the understanding of the adsorption and separation mechanism, as well as provide information on the relative stability of individual species.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM015847-30
Application #
2900455
Study Section
Special Emphasis Panel (NSS)
Project Start
1978-12-01
Project End
2001-03-31
Budget Start
1999-04-01
Budget End
2001-03-31
Support Year
30
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Northeastern University
Department
Type
Organized Research Units
DUNS #
039318308
City
Boston
State
MA
Country
United States
Zip Code
02115
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Li, Siyang; Nakayama, Tomoko; Akinc, Akin et al. (2015) Development of LC-MS methods for quantitation of hepcidin and demonstration of siRNA-mediated hepcidin suppression in serum. J Pharmacol Toxicol Methods 71:110-9
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Tummala, Seshu; Titus, Michael; Wilson, Lee et al. (2013) Evaluation of exogenous siRNA addition as a metabolic engineering tool for modifying biopharmaceuticals. Biotechnol Prog 29:415-24
Ni, Wenqin; Lin, Melanie; Salinas, Paul et al. (2013) Complete mapping of a cystine knot and nested disulfides of recombinant human arylsulfatase A by multi-enzyme digestion and LC-MS analysis using CID and ETD. J Am Soc Mass Spectrom 24:125-33
Ni, Wenqin; Bones, Jonathan; Karger, Barry L (2013) In-depth characterization of N-linked oligosaccharides using fluoride-mediated negative ion microfluidic chip LC-MS. Anal Chem 85:3127-35

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