Amino acid composition analysis of minute amounts of cysteine-containing proteins using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and 4-fluoro-7-nitro-2,1,3-benzoxadiazole in combination with HPLC.

The simultaneous determination of amino acid composition including cysteine of egg albumin, a model protein containing a/s cysteine residue, is reported. All the thiol groups of the cysteine residue(s) of egg albumin were labelled with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole, a fluorogenic reagent for thiol groups. The labeled egg albumin was hydrolyzed in 6N HCl at 110 degrees C for 24 h. The hydrolysate was lyophilized, derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, a fluorogenic reagent for amines, and subjected to HPLC. 18 derivatized amino acids including double labelled cysteine were separated within 90 min on a Nucleosil ODS column (150 mm X 4.6 mm i.d.; 5 microns), and detected at 530 nm (ex. 470 nm) in a range from 90 fmol (aspartic acid) to 1.3 pmol (cysteine) (S/N = 3). Composition ratios of amino acids of egg albumin were similar to theoretical values except for methionine, which would be destroyed under the present acid hydrolysis condition. Analytical methods for cysteine residues are reviewed, and the availability of fluorogenic reagents having the benzofurazan structure is also discussed.

[1]  E. Robinson,et al.  4-Thialaminine, a strongly basic chemical modification of cysteine. , 1972, The Journal of biological chemistry.

[2]  T. Toyo’oka,et al.  Application of a fluorogenic reagent, ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate for detection of cystine-containing peptides. , 1985, Journal of Biochemistry (Tokyo).

[3]  T. Toyo’oka,et al.  Further studies on the reaction of amines and proteins with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole , 1985 .

[4]  M. Friedman,et al.  The chromatographic determination of cystine and cysteine residues in proteins as s-beta-(4-pyridylethyl)cysteine. , 1970, The Journal of biological chemistry.

[5]  S. Moore,et al.  Automatic recording apparatus for use in the chromatography of amino acids. , 1958, Federation proceedings.

[6]  M. Ahnoff,et al.  Derivatization of secondary amino acids with 7-nitro-4-benzofurazanyl ethers , 1982 .

[7]  O. Blumenfeld,et al.  Reactions of N-ethylmaleimide with peptides and amino acids. , 1964, The Biochemical journal.

[8]  M. Friedman,et al.  New amino acids derived from reactions of epsilon-amino groups in proteins with alpha, beta-unsaturated compounds. , 1967, Biochemistry.

[9]  T. Liu,et al.  The stability of cysteine and cystine during acid hydrolysis of proteins and peptides. , 1970, The Journal of biological chemistry.

[10]  T. Toyo’oka,et al.  A novel fluorogenic reagent for thiols: ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate. , 1983, Analytical biochemistry.

[11]  L. Fothergill,et al.  The complete amino-acid sequence of hen ovalbumin. , 1981, European journal of biochemistry.

[12]  H. Gundlach,et al.  The nature of the amino acid residues involved in the inactivation of ribonuclease by iodoacetate. , 1959, The Journal of biological chemistry.

[13]  A. F. Bradbury,et al.  The use of 3-bromopropionic acid for the determination of protein thiol groups. , 1973, The Biochemical journal.

[14]  T. Toyo’oka,et al.  Fluorigenic reagents for primary and secondary amines and thiols in high-performance liquid chromatography. A review , 1984 .

[15]  R. Heinrikson.  The selective S-methylation of sulfhydryl groups in proteins and peptides with methyl-p-nitrobenzenesulfonate. , 1971, The Journal of biological chemistry.

[16]  K. Imai,et al.  Sensitive detection of amino acids in human serum and dried blood disc of 3 mm diameter for diagnosis of inborn errors of metabolism. , 1984, Journal of chromatography.

[17]  K. Imai,et al.  Liquid chromatographic determination of amino and imino acids and thiols by postcolumn derivatization with 4-fluoro-7-nitrobenzo-2,1,3-oxadiazole. , 1983, Analytical chemistry.

[18]  J. Holbrook,et al.  A peptide containing a reactive lysyl group from ox liver glutamate dehydrogenase. , 1969, The Biochemical journal.

[19]  S. Moore,et al.  The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. , 1963, The Journal of biological chemistry.

[20]  V. Švedas,et al.  The interaction of amino acids with ophthaldialdehyde: a kinetic study and spectrophotometric assay of the reaction product. , 1980, Analytical biochemistry.

[21]  R. W. Frei,et al.  The use of solvent segmentation in continuous-flow systems, and fluorescence labelling by derivatization , 1980 .

[22]  S. Fields,et al.  Sequence of chicken ovalbumin mRNA , 1978, Nature.

[23]  M. Weigele,et al.  Colorimetic amino acid analysis using fluorescamine. , 1975, Archives of biochemistry and biophysics.

[24]  S. Moore,et al.  Chromatography of amino acids on sulfonated polystyrene resins. , 1951, The Journal of biological chemistry.

[25]  B. Robberson,et al.  Modification of methionyl residues during aminoethylation. , 1967, Biochimica et biophysica acta.

[26]  T. Toyo’oka,et al.  Influences of metal ions on the reaction of amino and imino acids with fluorogenic reagents. , 1985, Analytical biochemistry.

[27]  T. Toyo’oka,et al.  New fluorogenic reagent having halogenobenzofurazan structure for thiols: 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole , 1984 .

[28]  T. Toyo’oka,et al.  High-performance liquid chromatography and fluorometric detection of biologically important thiols, derivatized with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F). , 1983, Journal of chromatography.

[29]  K. Imai,et al.  Fluorimetric determination of secondary amino acids by 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole , 1981 .

[30]  S. Simons,et al.  Ethanethiol: a thiol conveying improved properties to the fluorescent product of o-phthalaldehyde and thiols with amines. , 1977, Analytical biochemistry.

[31]  Sidney Udenfriend,et al.  Fluorescamine: A Reagent for Assay of Amino Acids, Peptides, Proteins, and Primary Amines in the Picomole Range , 1972, Science.

[32]  K. Imai,et al.  Pre-column labelling for high-performance liquid chromatography of amino acids with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole and its application to protein hydrolysates☆ , 1982 .

[33]  M. Roth.  Fluorimetric determination of free hydroxyproline and proline in blood plasma. , 1978, Clinica chimica acta; international journal of clinical chemistry.

[34]  H. Gundlach,et al.  The reaction of iodoacetate with methionine. , 1959, The Journal of biological chemistry.

[35]  T. Toyo’oka,et al.  Isolation and characterization of cysteine-containing regions of proteins using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and high-performance liquid chromatography. , 1985, Analytical chemistry.

[36]  W. Konigsberg,et al.  THE CHARACTERIZATION OF MODIFIED HUMAN HEMOGLOBIN. I. REACTION WITH IODOACETAMIDE AND N-ETHYLMALEIMIDE. , 1964, Journal of Biological Chemistry.

[37]  T. Toyo’oka,et al.  Determination of total captopril in dog plasma by HPLC after prelabelling with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F). , 1984, Journal of pharmaceutical and biomedical analysis.

[38]  T. Hofmann.  THE PURIFICATION AND PROPERTIES OF FRAGMENTS OF TRYPSINOGEN OBTAINED BY CYANOGEN BROMIDE CLEAVAGE. , 1964, Biochemistry.

[39]  J. J. Kolb,et al.  Methionine Studies. VII. Sulfonium Derivatives1 , 1945 .