Derivatization , proteins

The amidine bond is quite stable at acid pH however, it is susceptible to hydrolysis and cleavage at alkaline pH. Derivatized proteins may be assayed by amino acid analysis after acid hydrolysis without loss of imidate modifications. 

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.

Remove excess palmitic acid by chromatography on a column of Sephadex G-75. Use PBS, pH 7.4, containing 0.15 percent deoxycholate to perform the gel filtration. Collect the fractions containing derivatized protein, as monitored by absorbance at 280 nm. 

These inactivators typically have negligible reactivity toward cellular nucleophiles, in contrast to the classic affinity labels and the activated (escaped) form of suicide substrates (I ). However, all classes of irreversible inactivators - even in the ideal case of covalently labeling only their target enzymes - suffer from the possibility of eliciting an undesired immune response against the inactivator-derivatized protein following protein denaturation and degradation.1171... 

For SDS-protein complexes, the proteins can be derivatized with fluorescent dyes prior to the analysis. Derivatized proteins can be detected in the attomole (amol) level, and because the complex is resolved by sieving, multiple reaction products are detected as peak broadening instead of multiple peaks.19... 

The authors recognized that exhaustive acylation may lower PER of the protein due to unavailability of acylated lysine, and suggested that such derivatized proteins serve a functional role and not constitute a significant source of nutritive protein. 

Add the thiolated avidin-TCEP mixture dropwise to the dialyzed maleimide-derivatized protein solution with stirring Continue stirring for 1 h at room temperature, followed by stirring overnight at 4°C. 

It is essential that the procedure described in Section 3.1.2. be performed approx 24 h before the procedure described in Section 3.1 3, because the deprotected thiolated avidin and the maleimide derivative of the protein are unstable. Purification of the maleimide-derivatized protein by size exclusion chromatography can be performed more rapidly than dialysis, however, the former leads to dilution of the protein and a decrease in the yield of the conjugate. 

Nutritional Availability of Acylated Lysine and Proteins. The successful use of chemically derivatized proteins as food ingredients requires that they be digestible and nontoxic, and that the modified amino acid residues should be available nutritionally. Nutritional studies using modified food proteins are limited. The nutritional availability of several acylated lysines were studied by Bjarnason and Carpenter (105) and Mauron (106) and the results are summarized in Table III. The bioavailability of the acylated lysine varied significantly with the type of the acyl groups (see Table III). In addition acylated proteins (acetylated and succinylated) gave lower responses to the growth activity for the rats than equivalent supplements of unmodified proteins... 

DNP-derivatized proteins are detected on the nitrocellulose membrane as described in Protocol 11 except the primary antibody is a 1 200 dilution of rabbit anti-DNP antibodies (Sigma Chemical Co.). 

Succinylation reduced the isoelectric point of yeast proteins from pH A.5 to 4.0 and markedly improved their solubility in pH range 4.5 to 6. Heat denatured yeast proteins were facilely solubilized following succinylation (74). Succinylated yeast proteins were very stable to heat above pH 5, and remained soluble at temperatures above 80°C. As the degree of succinylation was increased the rate of precipitation of the derivatized protein increased in the neighborhood of the isoelectric point and much larger protein floes were obtained facilitiating their recovery (74). 

Na DH, Woo BH, Lee KC. Quantitative analysis of derivatized proteins prepared with pyridyl disulfide-containing cross-linkers by high-performance liquid chromatography. Bioconj. Chem. 1999 10 306-310. 

NMR Spectra. [ C]NMR analyses (1024 transients) were obtained using a Gemini 200 MHz spectrometer. Methylated protein samples were analyzed in an 8M urea, 90% DjO solution of 100 mM sodium phosphate which gave a pH meter reading of 8. For the aqueous reactions derivatized proteins were dialyzed against 10 mM HCl and lyophilized prior to the addition of 8M urea. Acetonitrile (30 1) was added to reference peak resonances. 

Other derivatives of tryptophan which may also be obtained after enzymic hydrolysis of derivatized proteins are those obtained by reaction with sulfenyl halides (Scoffone et al. 1968) and 2-hydroxynitro-benzyl bromide (Barman and Koshland 1967) ( 3.6.2). 

Pass the reduced derivatized protein through Sephadex G-25 equilibrated with 100 mM sodium acetate buffer, pH 4.5, containing 100 mM NaCl to remove excess reagent, oxidized reagent and pyridine-2-thionine. 

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