No abstract

The discovery of polypeptides and proteins with relevance to a particular biological state is complicated by their vast number and concentration range in most biological mixtures. Depletion methodologies are frequently used to remove the most abundant species; however, this removal not only fails significantly to enrich trace proteins, it may also nonspecifically deplete them due to their interactions with the removed high-abundance proteins. Here we report a simple-to-use methodology that reduces the protein concentration range of a complex mixture like whole serum through the simultaneous dilution of high-abundance proteins and the concentration of low-abundance proteins. This methodology utilizes solid-phase ligand libraries of immense diversity, generated by "split, couple, recombine" combinatorial chemistry, that are used for affinity-based binding to the proteins of a given mixture. With a controlled sample-to-ligand ratio it is possible to modulate the relative concentration of proteins such that many peptides or proteins that are undetectable by classical analytical methods become easily accessible. The reduction in the dynamic range of unfractionated serum is specifically described along with treatment of other proteomes such as extracts from Escherichia coli, chicken egg white and cell culture supernatant. Mono- and bi-dimensional electrophoresis (1-DE and 2-DE respectively) and surface-enhanced laser desorption/ionization-mass spectrometry (SELDI-TOF-MS) technology demonstrate the reduction in protein concentration range. Combining this approach with additional fractionation methods further increased the number of detectable species.

No abstract

C-reactive protein (CRP) is a phylogenetically conserved protein; in humans, it is present in the plasma and at sites of inflammation. At physiological pH, native pentameric CRP exhibits calcium-dependent binding specificity for phosphocholine. In this study, we determined the binding specificities of CRP at acidic pH, a characteristic of inflammatory sites. We investigated the binding of fluid-phase CRP to six immobilized proteins: complement factor H, oxidized low-density lipoprotein, complement C3b, IgG, amyloid ␤, and BSA immobilized on microtiter plates. At pH 7.0, CRP did not bind to any of these proteins, but, at pH ranging from 5.2 to 4.6, CRP bound to all six proteins. Acidic pH did not monomerize CRP but modified the pentameric structure, as determined by gel filtration, 1-anilinonaphthalene-8-sulfonic acid-binding fluorescence, and phosphocholine-binding assays. Some modifications in CRP were reversible at pH 7.0, for example, the phosphocholine-binding activity of CRP, which was reduced at acidic pH, was restored after pH neutralization. For efficient binding of acidic pHtreated CRP to immobilized proteins, it was necessary that the immobilized proteins, except factor H, were also exposed to acidic pH. Because immobilization of proteins on microtiter plates and exposure of immobilized proteins to acidic pH alter the conformation of immobilized proteins, our findings suggest that conformationally altered proteins form a CRP-ligand in acidic environment, regardless of the identity of the protein. This ligand binding specificity of CRP in its acidic pH-induced pentameric state has implications for toxic conditions involving protein misfolding in acidic environments and favors the conservation of CRP throughout evolution.

If endogenous TSE infectivity in RBCs binds to the ligands in the same proportion as brain-derived infectivity spiked into RBCs, the four most effective ligands would remove 3 to 4 log ID(50) per mL. A follow-up experiment is in progress to test whether endogenous blood-borne infectivity is also reduced.

Background: CRP exerts antipneumococcal function in mice if administered during the early stages of pneumococcal infection. Results: CRP loses such antipneumococcal function when its phosphocholine-binding pocket is blocked. Conclusion:The phosphocholine-binding pocket on CRP participates in antipneumococcal function of CRP during the early stages of infection. Significance: Remodeling of CRP may be required for successful treatment of mice during the late stages of infection.

No abstract

Plasmids in both Escherichia coli and Staphylococcus aureus contain an "operon" that confers resistances to arsenate, arsenite, and antimony(III) salts. The systems were always inducible. All three salts, arsenate, arsenite, and antimony(III), were inducers. Mutants and a cloned deoxyribonucleic acid fragment from plasmid p1258 in S. aureus have lost arsenate resistance but retained