Conjugate protein denaturants, effect

Effect of Protein Denaturants. Most of the conjugated enzymes we have prepared show greater resistance to inactivation than do the corresponding native enzymes when treated with protein... 

Proteins that have catalytic properties are called enzymes (i.e.. enzymes are biological catalysts of protein nature). Some enzymes have full catalytic reactivity per sc these are considered to be simple proteins because they do not hiive a nonprotein moiety. Other enzymes are conjugated proteins, and the nonprotcin structural components arc necc.ssary for reactivity. Occasionally, enzymes require metallic ions. Because enzymes are proteins or conjugated proteins, the general review of protein structural studies presented above in this chapter (e.g.. protein conformation and denaturation) is fundamental to the following topics. Conditions that affect denaturation of proteins usually have an adverse effect on the activity of the enzyme. 

Unfortunately, utilization of copper(i) to control the regioselectivity yields several problems. Although only catalytic amounts are required, copper can induce cytotoxic effects and protein denaturation, ° which can complicate bioconjugation by CuAAC for applications in vitro and in vivo. In addition, copper salts are often rather difficult to remove from the reaction products. Nevertheless, advantages like high selectivity, mild reaction conditions, and almost quantitative product formation have proven CuAAC highly beneficial for the preparation of site-specific peptide- and protein-polymer conjugates. 

Native, multi-subunit KLH also should not be frozen. Freeze-thaw effects cause extensive denaturation and result in considerable amounts of insoluble material. Commercial preparations of native KLH are typically freeze-dried solids that no longer fully dissolve in aqueous buffers and do not display the protein s typical blue color due to loss of chelated copper. The partial denatured state of these products often makes conjugation reactions difficult. 

Buffers are typically mixtures of solids and or a liquid and a solid. Examples of buffers include acetate buffer (50 mM at pH 4.0), phosphate buffer (lOOmM at pH 7.0), and 0.1 % TEA/0.1 % TFA. Since the use of buffers in RP separations is commonplace, basic knowledge of the solubility of each buffer component in the mobile phase is critical. Consider the case of phosphoric acid and its series of conjugate acid/base pairs as the buffer for acetonitrile/water mobile phase. Phosphate has negligible UV absorbance down to 200 nm (see Fig. 1.5denature proteins, and has three effective buffer regions around the pH values of 2, 7, and 12. These properties make phosphate buffers very attractive for many HPLC separations. 

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