Joseph R. Wagner

A rapid high-performance liquid chromatographic procedure for the simultaneous determination of methionine, ethionine, S-adenosylmethionine, S-adenosylethionine, and the natural polyamines in rat tissues

A method for the analysis of S-adenosyl-L-methionine (SAM) and S-adenosyl-L-ethionine (SAE) and their major metabolites by high-performance liquid chromatography is described. The procedure allows the simultaneous analysis of the natural polyamines, putrescine, spermidine, and spermine, and some of the major amino acids, methionine, tyrosine, and tryptophan. The uv absorbance at 254 nm is used for the determination of the SAM and SAE analogs, whereas the polyamines and amino acids are analyzed by fluorescence detection after postcolumn derivatization with o-phthalaldehyde. The method allows SAM and polyamine determinations by direct injection of the tissue extracts without prepurification. The procedure is applied to study the effects of DL-ethionine treatment on the SAM, SAE, methionine, and polyamine levels in various tissues of rats.

Estimation of the disulfide content of trypsin inhibitors as S-β-(2-pyridylethyl)-l-cysteine

Abstract Disulfide bonds in soybean trypsin inhibitor were reduced with tri- n -butylphosphine in 50% ethanol at pH 7.6. The generated SH groups were simultaneously alkylated with 2-vinylpyridine. Cystine residues were thus transformed to residues of S -β-(2-pyridylethyl)- l -cysteine. The extent of reductive alkylation was determined by both amino acid analysis and ultraviolet spectroscopy. The modified inhibitor was inactive against trypsin. It also lost resistance to proteolysis. Analogous results were obtained with partially reduced alkylated lima bean and ovomucoid trypsin inhibitors. These results show that S -2-pyridylethylation is a convenient method for measuring the half-cystine content of a protein.

Ion-exchange chromatography of sulfur amino acids on a single-column amino acid analyzer

Abstract Examination of the extent of production of the ninhydrin-colored derivative, Ruhemanns purple, under automated conditions of a single-column amino acid analyzer by several classes of sulfur-containing amino acids revealed a wide variation in the color factors relative to leucine. These ranged from 0.02 for the methyl ester of cysteine to 2.19 for D-homocystine. Color yields obtained by the manual ninhydrin reaction are generally lower than the corresponding values obtained on the amino acid analyzer. The elution positions ranged from 5.12 min for cysteic acid to 84.9 min for l -cystine dimethyl ester. The observed behavior of these compounds in the ninhydrin reaction is rationalized in terms of structural and electronic factors which they exhibit in reacting with ninhydrin to form the visible dye. Such an analysis should make it possible to predict ninhydrin color factors, and possibly also elution times, of structurally related compounds.

High-pressure liquid chromatography of steroidal alkaloids.

High-pressure liquid chromatography was used to separate the following steroidal alkaloids: tomatidine, solanidine, solasodine, rubijervine, veratramine and jervine. The method was used to prepare crystalline solanidine from a crude mixture of aglycones obtained from Solanum chacoense, and to separate radioactive solanidine from extracts of potato plants fed with [4-C]cholesterol.