Reactions of Native Proteins with

Herriott, R.M. (1947) Reactions of native proteins with chemical reagents. Adv. Protein Chem. 3, 169. 

Reactions of Native Proteins with Chemical Reagents Roger M. Herriott... 

REACTIONS OF NATIVE PROTEINS WITH CHEMICAL REAGENTS 193... 

Bradbury and Norton (1975), on the basis of the model building studies by Browne et al. (1969) and by Warme et al. (1974), were able to make assignments of specibc resonances in the proton NMR spectrum of bovine a-lactalbumin to the three His residues. These resonances, after reaction of the protein with iodoacetate under conditions that were nearly specific for the His residues, disappeared from the native frequency positions, but did so in a differential manner, consistent with differences in the degrees of exposure to the solvent. This was predicted by the model building studies, particularly those by Browne et al., which were subsequently confirmed by X-ray analysis. Thus, His-68 is the most exposed His-32, being involved in a helical region (according to Browne et al., but not to Warme et al.), is less exposed, while His-107 is the least exposed. 

There are other substrates for the E. coli Met(0) peptide reductase, one of which is Met(0)-a-l-PI. The native protein is the major serum elastase inhibitor that functions by forming a binary complex with elastase which inhibits its activity. Met(0)-a-l-PI, on the other hand, which can be formed by treatment of the protein with TV-chlorosuccinimide, cannot form a complex with elastase and therefore is not able to inhibit elastase activity117,118. Table 6 shows, however, that when Met(0)-a-l-PI is incubated in the presence of Met(0)-peptide reductase and dithiothreitol the protein regains its ability to form a complex with elastase and inhibit elastase activity119. Similar to results found with Met(0)-L12 reduced thioredoxin could replace the dithiothreitol as reductant in the enzymatic reaction. 

The detection and determination of deteriorated proteins is probably one of the most difficult problems in protein chemistry today. The main reason is that it is usually easier to determine native properties, particularly when those properties include a biochemical function, than it is to determine a deteriorated and inactive protein. In addition, there are almost always no standards to use as a measuring stick for the deteriorated protein. When the deteriorative reactions are extensive and more than a fraction (30-40%) of the protein undergoes deterioration, the deterioration can frequently be monitored by determining the amount of native protein remaining, providing, of course, that the deteriorated protein does not interfere with the determination of... 

Fig. 4. Scheme of NCL. The mechanism allows the straightforward preparation of small proteins with native backbone structures from fully unprotected synthetic peptide building blocks. The initial tran -thioesterification step includes the chemo-selective reaction between one peptide with a C-terminal a-thioester group (peptide 1) and second peptide with an N-terminal cysteine residue (peptide 2). Generated thio-ester-linked intermediate spontaneously rearranges to form a native peptide bond at the site of ligation. 

The characterization of proteins and their derivatives is outside the scope of this monograph. It is necessary to emphasize, however, that modification of native proteins frequently gives rise to complex mixtures of products. The complexity of the situation is not inunediately apparent solely from the stoichiometry of the modification reaction. The now classical case of the reaction of native bovine pancreatic ribonuclease A with iodoacetic acid at pH 5.5 may be cited in this context. One carboxymethyl group is introduced per molecule of... 

Nitration of tyrosyl residues with tetranitromethane (TNM) is now one of the most frequently attempted modification reactions for native proteins (see Riordan and Sokolovsky 1971 for a review and references). The mechanism of this reaction has been examined by Bruice et al. (1968), who propose the following scheme ... 

Much information about the relationship between the stracture of a protein and its function has been determined by site-specific mutagenesis, a technique that replaces one amino acid of a protein with another. For example, when Asp 102 of chymotrypsin is replaced with Asn 102, the enzyme s abihty to bind the substrate is unchanged, but its ability to catalyze the reaction decreases to less than 0.05% of the value for the native enzyme. Clearly, Asp 102 must be involved in the catalytic process. We have seen that its role is to position histidine and use its negative charge to stabilize histidine s positive charge. 

The spectrophotometric evidence reviewed above for the binding of a proportion of the phenolic hydroxyl groups of the tyrosine residues of native proteins is supported by work on the action of tyrosinase on proteins. Sizer (1946) found that this enzyme oxidizes the tyrosine residues in native trypsin, pepsin, chymotrypsin, casein, peptone, insulin, and hemoglobin. Native ovalbumin, human and bovine serum albumin, tobacco mosaic virus (nucleoprotein), human y- and bovine /3-globulins, and bovine fibrinogen are not susceptible to tyrosinase, but become so after tryptic digestion. It was shown (Sizer, 1947) that for the proteins which are oxidized by tyrosinase in the native state, the observed reaction does indeed occur with the intact proteins and does not require preliminary degradation to tyrosine peptides or free tyrosine. The kinetics of the oxidation of tyrosine by tyrosinase have been studied spectropho-tometrically (Mason, 1948 etc.). 

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