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Synthesis, Purification, and Properties of a Semisynthetic Ribonuclease S Incorporating 4-Fluoro-l-histidine at Position 12
Journal article   Open access   Peer reviewed

Synthesis, Purification, and Properties of a Semisynthetic Ribonuclease S Incorporating 4-Fluoro-l-histidine at Position 12

Ben M. Dunn, Carlo DiBello, Kenneth L. Kirk, Louis A. Cohen and Irwin M. Chaiken
The Journal of biological chemistry, v 249(19), pp 6295-6301
10 Oct 1974
DOI: https://doi.org/10.1016/S0021-9258(19)42252-7
PMID: 4420810
url
https://doi.org/10.1016/s0021-9258(19)42252-7View
Published, Version of Record (VoR) Open
url
https://doi.org/10.1016/S0021-9258(19)42252-7View
Published, Version of Record (VoR) Open

Abstract

The NH2-terminal pentadecapeptide of ribonuclease was synthesized with 4-fluoro-l-histidine (4-F-His) in place of the naturally occurring histidine at position 12. This peptide, [4-F-His 12]synthetic-(1-15), serves as an active site analogue of the native NH2-terminal eicosapeptide, RNase-S-(1-20), in that it forms a stable, noncovalent complex with the native ribonuclease fragment of residues 21 through 124, RNase-S-(21-124). A functional purification scheme has been used to prepare purified synthetic peptide in two steps. Purified semisynthetic ribonuclease S analogue complex was obtained by sulfoethyl-Sephadex chromatography of a mixture of crude, solid phase-derived peptide and native RNase-S-(21-124). Isolated [4-F-His 12]synthetic-(1-15) peptide was obtained from the purified complex after dissociation in 50 % acetic acid and gel permeation chromatography. The substitution of fluorine in the 4 position of the imidazole ring of histidine 12 does not disrupt the association of the synthetic peptide to native RNase-S-(21-124) as demonstrated by the binding constant measured in competition with native RNase-S-(1-20). Several other conformational properties of the analogue complex also are similar to those of the native RNase-S complex. However, the complex of [4-F-His 12]-synthetic-(1-15) and RNase-S-(21-124) is devoid of catalytic activity, a result attributed to the perturbation in the acid dissociation constant of the imidazole side chain by fluorine substitution.

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