Characterization of guanidinated cytochrome c by carbon-13 nuclear magnetic resonance spectroscopy

Abstract

in which the longest decay component could be unambiguously determined, these measurements represent an important check on the validity of the conclusions drawn from the anti-DNS studies. Furthermore, the measurements were carried out on an antibody population with a different specificity. Thus, Fab segmental flexibility is probably generally present in IgG molecules. 19 homoarginine residues are distributed over a range of about 1 ppm and include two discrete single guanido carbon resonances. Distinct changes in the 13C NMR spectrum are observed upon complexation of guanidinated ferricytochrome c with ferrihexacyanide, upon conversion of the guanidinated ferricytochrome c to its alkaline isomer, and upon reduction to guanidinated ferrocytochrome c. Analysis of the changes in the visible absorbance spectrum accompanying guanidination of ferricytochrome c with O-methylisourea indicates that 18 lysine residues are guanidinated about 3 times more rapidly than the single remaining lysine residue, most likely lysine-79. The guanido carbon of this residue was preferentially enriched with l3C-enriched O-methylisourea. Analysis of the Ferricytochrome c is known to form discrete complexes which facilitate electron exchange with electron acceptors such

Soybean trypsin inhibitor (Kunitz) and its complex with trypsin. Carbon-13 nuclear magnetic resonance studies of the reactive site arginine

“…When L-arginine is incorporated into position 63 of STI, the Cl shifts -0.24 ppm (upfield); upon subsequent formation of the STI-trypsin complex, the Arg63 Cf shifts +0.23 ppm (downfield). These environmental shifts are comparable to those reported by Oldfield et al (1975) for several small proteins but are smaller than some found recently for [13C']-L-homoarginine and pC*]-L-arginine in other proteins (Stellwagen et al, 1977;Cocco et al, 1977). In the latter studies, a paramagnetic heme group (Stellwagen et al, 1977) or a large aromatic nucleotide ligand (Cocco et al, 1977) was present.…”

Ionization of tyrosine and lysine residues in native and modified horse cytochrome c

“…The NE-acetimidyl derivatives of [Hse65lcytochrome c were prepared by the method of Wallace & Offord (1979). NE-Amidino-cytochrome c was prepared in accordance with published procedures (Hettinger & Harbury, 1964;Stellwagen et al, 1977) by the reaction of 0methylisourea hydrogen sulphate with cytochrome c (type III). The modified protein was purified in the same manner as N'-acetimidyl-cytochrome c. Amino acid analysis of the major product indicated that the reaction was > 98% complete.…”

Methyl motions in 13C-methylated concanavalin as studied by carbon-13 magnetic resonance relaxation techniques

“…The average chemical shift observed for each of these envelopes is identical with those observed in a SDS-denatured sample, indicating that these lysines are all surface residues and readily accessible to the solvent. Stellwagen et al (1977) and Cocco et al (1978) have observed relationships between the chemical shift and segmental motion (as measured by T{s) of surface-exposed residues; i.e., a residue experiencing decreased motion will also be chemically shifted the furthest from the position of the solvent-exposed residues. We have been unable to detect any significant differences in the spin-lattice relaxation times of the shoulders of these two envelopes which suggests that all residues which comprise each envelope do indeed relax similarly.…”

“…We have been exploring ways which may be used to examine these protein-cell interactions using high-resolution NMR techniques; one approach is to label surface residues on Con A with NMR-active nuclei which might be sensitive to interactions occurring between the protein and a cell surface. Stellwagen et al (1977) converted the surface lysines of cytochrome c to homoarginine residues by using 90% [13C]-<9methylisourea and showed that the resulting 13C resonances were sensitive to the chemical environment and segmental motion experienced by each homoarginine residue. Biological methods have been successfully used to label dihydrofolate reductase with [13C]arginine (Cocco et al, 1978).…”