Determination of chemical properties of individual histidine and tyrosine residues of concanavalin A by competitive labeling with 1-fluoro-2,4-dinitrobenzene.
A selective peptide-mapping procedure was devised to purify peptides containing histidine or tyrosine residues from proteolytic digests of concanavalin A (Con A). The protein was modified with maleic anhydride followed by 1-fluoro-2,4-dinitrobenzene (Dnp-F) and then digested with thermolysin. The resulting labeled peptides were separated by high-performance liquid chromatography, and the Dnp-histidine and Dnp-tyrosine peptides were identified by their spectral characteristics. From their amino acid compositions, the labeled peptides could all be assigned within the known sequence. Peptides representing five of the six histidines and all seven tyrosines were obtained. With the same peptide-mapping procedure, the chemical properties (pK and reactivity) of these residues were determined. Samples of concanavalin A at various pH values were labeled with trace amounts of [3H]Dnp-F, in the presence of Gln-Gly as an internal standard. To each sample was added an aliquot of a mixture of [14C]Dnp-Gln-Gly and [14C]Dnp-maleyl-Con A. Portions of each sample were removed, [14C]Dnp-Ala-Ala and epsilon-[14C]Dnp-lysine were added, and the mixtures were hydrolyzed. The various Dnp amino acid derivatives were purified by HPLC. The remainder of each [3H]Dnp sample was maleylated, dinitrophenylated, and digested with thermolysin and separated by HPLC as above. From the 3H/14C ratios of the Dnp amino acid derivatives and the Dnp peptides relative to the ratio of the internal standard, pK and reactivity data were obtained for (a) the average behavior of the lysine, histidine, and tyrosine residues and (b) the individual behavior of the N-terminal alanine residue and the five histidine and seven tyrosine residues in the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
G E
GE
Jackson
N M
NM
Young
D000595:Amino Acid Sequence; D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid; D003208:Concanavalin A / Q000302:isolation & purification; D004139:Dinitrofluorobenzene; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D010446:Peptide Fragments / Q000302:isolation & purification*; D014443:Tyrosine
D002250:Carbon Radioisotopes; D010446:Peptide Fragments; D003208:Concanavalin A; D014443:Tyrosine; D006639:Histidine; D004139:Dinitrofluorobenzene