muramidase and 3-bromopropylamine

We have previously shown that 3-bromopropylamine offers several advantages over other alkylating reagents in the modification and subsequent identification of cysteine residues by protein sequencing. We describe here simple on-sequencer procedures for alkylating cysteines in proteins which employ the reduction of cystines in proteins with tri-n-butylphosphine and concomitant alkylation of the resulting cysteines with 3-bromopropylamine. Addition of an aqueous acetone wash to a modified reaction cycle on the Applied Biosystems 477A sequencer removes excess 3-bromopropylamine. As a result, very little background in the first step of the sequence analysis is seen. Under these conditions, cysteines are readily modified and identified during sequencing. Moreover, very little preview of the next amino acid is observed, which indicates that the N-terminal amino acid is not appreciably alkylated by 3-bromopropylamine. On-sequencer methods have been developed for proteins spotted onto glass fiber filters and proteins electroblotted onto polyvinylidene difluoride membranes.

Topics: Alkylation; Amino Acid Sequence; Cysteine; Indicators and Reagents; Muramidase; Online Systems; Oxidation-Reduction; Phenylthiohydantoin; Phosphines; Propylamines; Proteins

A new reagent for the routine identification of cysteine residues during protein sequencing is described. This method employs 3-bromopropylamine to alkylate cysteines in proteins after reduction with dithiothreitol. Upon sequencing of the protein on an Applied Biosystems 477A protein sequencer, the aminopropylcysteine residue yields a phenylthiohydantoin (PTH) derivative which elutes reproducibly at a unique position immediately after PTH-leucine; baseline resolution is achieved without modification of the PTH analyzer gradient. Unlike PTH-pyridylethylcysteine, the relative elution position of the aminopropylcysteine PTH derivative is not affected by changes in the ionic strength of the analyzer solvents. In addition, the previewing of the next amino acid which is observed in proteins modified with 4-vinylpyridine does not occur in aminopropylated proteins. Preparation of alkylated proteins for electroblotting onto polyvinylidene difluoride (PVDF) membranes and methods for desalting alkylated proteins immobilized on precoated glass fiber filters or PVDF membranes are also described.

Topics: Alkylating Agents; Alkylation; Cysteine; Metallothionein; Muramidase; Phenylthiohydantoin; Propylamines; Proteins; Sequence Analysis