Reaction detector system for the simultaneous monitoring of primary amino groups and sulfhydryl groups in peptides eluted by high-performance liquid chromatography.
A reaction detector system is described which can be used for simultaneous analysis of primary amino groups and sulfhydryl groups in peptides separated by high-performance liquid chromatography. By use of an automatic split valve system, well-defined portions of the effluent are diverted alternately into one of the two reaction detectors, based on the ninhydrin and Ellman assay, respectively. In the latter case the dithioerythritol included in the elution buffers for thiol preservation is eliminated with arsenite. For selective recovery of eluted peptides the amino/thiol group analyser can easily be converted into a high-yield monitoring system compatible with volatile, UV-opaque gradient buffers. In this version an automatic sample valve is used for the thiol assay, removing picomole amounts of material from the effluent destined for collection. In preparative applications there is a progressive net gain in sensitivity with increasing proportion of collected material. Under conditions of optimum recovery ca. 90% of the material could be recovered at a total consumption in the two detectors of less than 100 pmol. Experimental conditions are described which would allow for the additional identification of tryptophan, tyrosine and lysine-containing peptides. The monitoring system may also be used without modification for the assessment of key residues in polypeptides and proteins.
D000588:Amines / Q000032:analysis*; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D009555:Ninhydrin; D010455:Peptides / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000032:analysis*
D000588:Amines; D010455:Peptides; D013438:Sulfhydryl Compounds; D009555:Ninhydrin