ABSTRACT
A high-concentration fluid may be defined as one in which the distance between the van der Waals surfaces of the molecules is on the same order of magnitude as the size of the molecules. Many biological fluids meet this definition of being concentrated. High concentration protein solutions are of commercial interest, too. In the biopharmaceutical industry, there is a desire to produce high concentration (>100 mg/ml) protein formulations. The goal of this paper is to provide an energetic framework for understanding the functional properties (e.g., solubility, stability and viscosity) of high concentration solutions. Using this framework not only provides a simple, clear view of protein behavior at high concentrations, it also provides insights into how modifications to the protein or the solvent affect solution behavior. Thus, the paper provides a starting point for the rational manipulation of protein solubility, viscosity and stability.
Subject(s)
Proteins/chemistry , Solutions/chemistry , Algorithms , Chemistry, Pharmaceutical , Electrochemistry , Protein Binding , Protein Stability , Solubility , Thermodynamics , ViscosityABSTRACT
The fang-like jaws of the marine polychaete Nereis virens possess remarkable mechanical properties considering their high protein content and lack of mineralization. Hardness and stiffness properties in the jaw tip are comparable to human dentin and are achieved by extensive coordination of Zn (2+) by a histidine-rich protein framework. In the present study, the predominant protein in the jaw tip, Nvjp-1, was purified and characterized by partial peptide mapping and molecular cloning of a partial cDNA from a jaw pulp library. The deduced amino acid sequence revealed an approximately 38 kDa histidine-rich protein rich in glycine and histidine (approximately 36 and 27%, respectively) with no well-defined repetitive motifs. The effects of pH and metal treatment on aggregation, secondary structure, and hydrodynamic properties of recombinant Nvjp-1 are described. Notably, Zn treatment induced the formation of amyloid-like fibers.
Subject(s)
Polychaeta/chemistry , Protein Structure, Secondary , Amino Acid Sequence , Amyloid/chemistry , Animals , Cloning, Molecular , Gene Library , Glycine/chemistry , Histidine/chemistry , Hydrogen-Ion Concentration , Jaw/chemistry , Microscopy, Atomic Force , Molecular Sequence Data , Peptide Mapping , Recombinant Proteins/biosynthesis , Solubility , Spectroscopy, Fourier Transform Infrared , Zinc/chemistryABSTRACT
For nearly 50 years David Yphantis has helped advance analytical ultracentrifugation, promoted rigor in the thermodynamic analysis of biochemical data and encouraged students and colleagues to look for the deepest possible understanding of science. This article, written by five of Dave's students, presents some of the impressions he has made over the years.
Subject(s)
Ultracentrifugation/methods , Biochemistry/methods , Biophysics/methods , History, 20th Century , Humans , Macromolecular Substances , Thermodynamics , Ultracentrifugation/history , Ultracentrifugation/instrumentation , Ultracentrifugation/statistics & numerical dataABSTRACT
Analytical ultracentrifugation (AUC) is a powerful means of characterizing the solution behavior of molecules. Sedimentation velocity analysis, the preferred AUC technique for characterizing complex systems, has higher resolution, broader applicable range and fewer solute/solvent limitations than gel-permeation chromatography. The technique is simple to perform and should become a mainstay for target identification, target validation, lead optimization, formulation in drug development and QA/QC. Recent studies have used AUC to characterize the binding stoichiometry and binding sites of an anti-tumor agent; of a hemoglobin-stabilizing protein, and of a fibril growth inhibitor, and to assess the causes of protein aggregation. The recent addition of fluorescence to the existing absorbance and interference detectors dramatically extends the flexibility of analytical ultracentrifugation.: