Alkylamine derivatives of cytochrome c

Abstract

For investigations of the functional roles of the lysine residues of cytochrome c, analogues in which these residues are modified without charge loss are highly desirable. The 19 lysine residues of the horse heart protein have been modified by reductive alkylation. Two analogues were prepared, using formaldehyde and acetone as the dialkylating and monoalkylating reagent respectively. Modification was shown to be clean and quantitative. Characterisation of the alkylamine derivatives by physicochemical measurements and biological activity determinations was carried out. The potential of these analogues in structure/function studies of cytochrome c is discussed. It is illustrated by their use, in conjunction with other lysine‐modified derivatives, to investigate the extent to which surface charge determines redox potential, and to study the physicochemical changes that accompany rising pH. In the latter case the observed phenomena are not as closely correlated as previously thought, suggesting that there is a complex set of rearrangements of structure underlying the functional changes. The data confirm that modification of the lysine residues influences these changes. These residues participate in numerous surface intramolecular links, so the lack of correlation may be explained if each of the changing parameters were under the influence of a different set of residues. However, neither a lysine residue, nor a histidine residue directly displaces methionine from the sixth coordination position of the haem iron at alkaline pH.

NMR study of the alkaline isomerization of ferricytochrome c

“…The pKa value of cytochrome c in D20 at 27°C was calculated as 9.5 + 0.1 from the forward and reverse rate constants, kf and kr, using the expression: pK~ = pH* + log(kr/kf); the same value was derived from the integrated areas of NMR peaks for the native and basic forms. This calculated pKa value agrees with that measured by others [1][2][3][4][5][6][7][8][13][14][15][16][17]22]. To calculate activation parameters for this process, rate constants were measured at six different temperatures ranging from 27 to 55°C.…”

“…One of the best studied of these changes is the isomerization that occurs with an apparent pKa of approx. 9.5 to give an alkaline form of cytochrome c. The structure of the alkaline form is still unclear, however, with a protein lysine [2], protein histidine [3] or hydroxide [4] proposed as an axial ligand replacing the methionine [5].…”

Glycans on Secretory Component Participate in Innate Protection against Mucosal Pathogens

“…Radiolabeling of C. difficile Toxin A-Toxin A was labeled by alkylation-reduction based on the protocol of Wallace and Corthésy (21). Briefly, 50 g of toxin A was dissolved in 200 l of 0.2 M sodium borate buffer (pH 9.0) containing 10 g of NaBH 4 […”

Chemistry in Proteomics: An Interplay between Classical Methods in Chemical Modification of Proteins and Mass Spectrometry at the Cutting Edge

“…This reaction yields mainly the ε-N,N-dimethyl derivative of lysine and the ε-N-methyl analogue as a byproduct (Means and Feeney, 1968). The protocol of this method was later improved (Jentoft and Dearborn, 1983;Wallace and Cortésy, 1987), allowing all the 19 lysine residues involved in horse cytochrome c to be fully dimethylated as indicated by ESI-MS as well as the corresponding number of 13 C NMR signals due to the labeled ε-N,N-di-[ 13 CH 3 ]methyl-amino group of lysine (Moore et al, 1998).…”