RNase carboxymethylated

Affinity chromatography was carried out on columns prepared with lightly carboxymethylated chitin, which is known to be a poor substrate for lysozyme. Both native lysozyme and regenerated 13-105 were bound to the column at pH 7 and eluted at pH 3. As controls, the basic proteins cytochrome c and pancreatic RNase A, as well as concanavalin A and a-amylase, were not bound from the same solvent at pH 7. These findings constitute a third line of evidence for formation of native-like structure in regenerated 13-105. 

Haloacetates. In alkaline solution lysine residues can be alkylated in the presence of iodo- or bromoacetate ions (118). Both mono- and di-carboxymethyl derivatives can be formed. Some of the characterized derivatives are listed at the end of Table VI. Number 25, t-CM-Lys-41-RNase, shows a very low activity when measured by a step 2 assay employing C > p as substrate. This same compound is active in the depolymerization of 5S RNA (119) but the evidence presented to show that it is not the result of contamination with native RNase-A can be interpreted to suggest the opposite. 

In a more detailed study, Crestfield et al. 159) found that the reaction with iodoacetate at pH 5.5 produced two monosubstituted derivatives. The major inactive product was l-carboxymethyl-His-119-RNase,... 

When RNase A is treated with iodoacetate (ICH2CO2 ), the two major products obtained are carboxymethylated derivatives of His 12 and His 119. Both of these enzymes are severely inhibited, which suggests that both His 12 and His 119 are important in the active site. The enzyme also is completely inhibited by the reaction of Lys 41 with fluorodinitrobenzene. 

Fig. 8.12. Urea gradient electrophoresis of (A) native and reduced carboxymethylated bovine RNase A (0.05 M Tris-acetate buffer, pH 4.0) (B) bovine serum albumin (Tris-borate EDTA buffer, pH 8.6). The linear gradient of 0-8 M urea was superimposed on the inverse linear gradient of 15-11% acrylamide (courtesy of Creighton, 1979a).

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