Protein reaction

M Karplus. Aspects of protein reaction dynamics Deviations from simple behavior. J Phys Chem B 104 11-27, 2000. 

Figure 9.3 Schematic illustration of the electrophoretic transfer of proteins in the chromatophoresis process. After being eluted from the HPLC column, the proteins were reduced with /3-mercaptoethanol in the protein reaction system (PRS), and then deposited onto the polyacrylamide gradient gel. (PRC, protein reaction cocktail). Reprinted from Journal of Chromatography, 443, W. G. Button et al., Separation of proteins by reversed-phase Mgh-performance liquid cliromatography , pp 363-379, copyright 1988, with permission from Elsevier Science.

Enzyme or Protein Reaction Catalyzed or Function Comment... 

Verveer, P. J., Squire, A. and Bastiaens, P. I. (2001). Improved spatial discrimination of protein reaction states in cells by global analysis and deconvolution of fluorescence lifetime imaging microscopy data. J. Microsc. 202, 451-6. 

Figure 3.1 Two essential steps of chemical reaction of formaldehyde (HCHO) with nucleic acid exemplified by adenine that are similar to formaldehyde-protein reactions, (a) Addition reaction as the first step, resulting in a methylol derivative, methylol adenylic acid (b) Second step is a condensation reaction, a stable product methylene-bis-adenylic acid is derived between the methylol derivative and another adenine. Reproduced with permission from Shi et al.,AIMM 2001 9 107-116. 

Metz B, Kersten GFA, Hoogerhout P, et al. Identification of formaldehyde-induced modifications in proteins reactions with model peptides. J. Biol. Chem. 2004 279 6235-6243. 

Dissolve mono(lactosylamido) mono(succinimidyl)suberate in dry DMF to prepare a concentrated solution from which an aliquot may be taken and added to a final aqueous reaction medium. The compound is extremely soluble in DMF, and solutions of 100 mg/ ml may be prepared. The use of dry solvent is essential to prevent hydrolysis of the NHS ester. However, make only enough of this stock solution so that a small amount added to the protein reaction will provide the appropriate molar excess desired for the modification reaction. 

Reactions done with NHS-PEG -biotin compounds typically are done with the reagent in molar excess over the amount of protein being modified. The efficiency of the reaction is dependent on the concentrations of reactants and the solvent exposed area of the amine groups on the protein. Reactions done with a 10-fold molar excess of NHS-PEG -biotin usually will result in at least 2-3 biotin labels per protein, while doubling the molar excess should provide 4-6 biotinylations. The optimal number of biotin groups added to a particular protein should be determined experimentally to provide the best performance in the intended application. 

Figure 19.19 An Ellman s assay may be done to determine the maleimide activation level of SMCC-derivatized proteins. Reaction of the activated carrier with different amounts of 2-mercaptoethanol results in various levels of sulfhydryls remaining after the reaction. Detection of the remaining thiols using an Ellman s assay indirectly indicates the amount of sulfhydryl uptake into the activated carrier. Comparison of the Ellman s response to the same quantity of 2-mercaptoethanol plus an unactivated carrier indicates the absolute amount of sulfhydryl that reacted. Calculation of the maleimide activation level then can be done.

Masri, M.S., and Friedman, M. (1988) Protein reactions with methyl and ethyl vinyl sulfones./. Protein Chem. 7, 49-54. 

Results obtained for the blue Cu protein plastocyanin are considered here in detail as illustrative of different approaches yielding relevant information. The reduction of plastocyanin PCu(II) with cytochrome c(II) is also considered as an example of a protein-protein reaction. 

The aim in solution studies on metalloprotein is to be able to say more about intermolecular electron transfer processes, first of all by studying outer-sphere reactions with simple inorganic complexes as redox partners. With the information (and experience) gained it is then possible to turn to protein-protein reactions, where each reactant has its own complexities... 

As we pointed out earlier, the H subunit catalyses the ferroxidase reaction, which occurs at all levels of iron loading, but decreases with increasing amounts of iron added (48-800 Fe/ protein). Reaction (19.8) catalysed by both FI- and L-chain ferritins, occurs largely at intermediate iron loadings of 100-500 Fe/protein. Once nucleation has taken place, the role of the protein is to maintain the growing ferrihydrite core within the confines of the protein shell, thus maintaining the insoluble ferric oxyhydroxide in a water-soluble form. 

While the results of this work are encouraging, it is clear that the structural definition of mutant proteins of this type is critical to development of rational interpretation of the results if for no other reason than that the structural perturbation introduced is presumably greater than for simple point mutations. Moreover, it would be particularly interesting to compare the functional properties of mutants compared in this manner in assays involving protein-protein reactions relevant to the species of cytochrome c on which the mutagenesis is based. For example, comparison of the activities of wild-type yeast cytochrome c with that of a loop-insertion mutant modelled on a photosynthetic cytochrome c in the reaction with the photosynthetic reaction center could help define the structural elements involved in the cytochrome c binding domain for the reaction center. 

In protein-protein reactions, the donor-acceptor distance is determined by the structure of the reacting proteins, and the way(s) in which they bind and interact. For example, it is generally believed that cytochrome c binds to its reaction partners at or near the exposed heme edge, in order to minimize the reactant distance and thereby maximize the rate. The redox active centers of most proteins are sufficiently buried that the large protein imposed distances provide low intrinsic reactivity for the proteins with respect to exogenous... 

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