Abstract
The Reactivities of the Histidine Residues at the Active Site of Ribonuclease toward Halo Acids of Different Structures
Highlights
Reacts at pH 5.5 with iodoacetic acid, alkylation occurs at either imidazole nitrogen 3 of histidine-12 or at imidazole nitrogen 1 of histidine-119
The data presented in this communication extend these observations and indicate that, under the same conditions, reaction with a series of unbranehed LY-and,&bromo acids ranging in length from 3 to 6 carbon atoms leads to alkylation at the same sites
The alkylation of ribonuclease A at pH 5.5 and 25” by a series of unbranched CY-and P-halo acids ranging in chain length from 3 to 6 carbon atoms has been investigated
Summary
Ribonuclease A-Lyophilized, phosphate-free ribonuclease A (RAF-6045) was purchased from Worthington BiochemicalCorporation. The ribonuclease A used in kinetic studies was obtained by purification of 500-mg portions of the commercial material on a column (4 x 34 cm) of sulfoethyl-Sephadex C-25 as described by Crestfield, Stein, and Moore (5). To permit repeated reuse of the same column, the aggregates in the commercial enzyme were first removed by passing a solution containing 0.5 g of protein in 5 ml of Hz0 through a column (0.9 x 5 cm) of the same ion exchanger equilibrated with the same buffer (cf Reference 6). The purified enzyme eluted from the preparative column was concentrated to 5 ml by rotary evaporation and was obtained in 0.1 M acetate buffer at pH 5.5 by gel filtration of the concentrated solution on a column (2 x 34 cm) of Sephadex G-25 (bead form) equilibrated with this buffer. The yield was about 300 mg calculated on an anhydrous basis
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