(16k)Cysteine (Cys) is typically difficult to identify when sequencing proteins using the Edman degradation procedure because its phenylthiohydantoin (PTH) derivative is unstable, losing H2S. Derivatives of Cys in which the sulfhydryl group has been alkylated to form a thioether are stable, however. For sequencing purposes, the Cys sulfhydryl group is usually alkylated by reaction with 4-vinyl pyridine, a reaction originally devised by Friedman and coworkers (I) for determination of Cys during amino acid analysis. Previously, Cavins and Friedman (2) found that Cys in proteins was alkylated by ,B-addition to the vinyl groups of acrylonitrile, acrylamide, and methyl acrylate.
Recently, I have observed that proteins blotted onto polyvinylidine difluoride membranes (e.g. immobilon P) from polyacrylamide gels often contain Cys residues that have been alkylated by acrylamide remaining in the gel after polymerization, and that this alkylated derivative (S-beta-propionamido Cys) is quite useful for identifying Cys within a protein sequence. It is well separated from other PTH amino acid residues using standard reverse phase HPLC methods and gives a sharp, narrow peak that is easily identified. In fact, during sequencing on the Porton 2090 sequencer, it is easier to identify than is the PTH derivative of s-,beta-(4-pyridylethyl) Cys, which is formed from Cys residues alkylated with 4-vinyl pyridine.
Table I shows the retention times obtained for the 19 commercially available PTH amino acids as well as for the acrylamide derivative of PTH Cys.
(16k)
HPLC chromatograms obtained during sequencing also contain large peaks at about 16.5 minutes (due to a side product formed from dithiothreitol contained in Porton reagent S1, which is ethyl acetate), and at 19.1 minutes (due to diphenylthiourea). My experience has been that the PTH derivative of S-,l~-(4-pyridylethyl) Cys elutes either with the 16.5 min. peak or just after it, where it can be confused with the PTH-Pro peak at about 16.9 minutes.
Besides occurring as an accidental side product from reaction of Cys in proteins with residual acrylamide in gels, S-beta-propionamido Cys can easily be generated intentionally by reacting reduced, denatured proteins with acrylamide under mildly alkaline conditions.
(16k)
Table II shows the results of N-terminal sequence analysis performed on myotoxin a, a small snake venom protein (MW = 4823) after reduction by heating at 65deg.C for 30 minutes in the presence of 100 mM DTT and 2% SDS. The 100 ~I sample was then diluted to 1 mL and concentrated back to 100 ul by centrifugation in a centrifree ultrafiltration unit (MW cutoff = 1000). Acrylamide was then added to give a final concentration of 2 M in a total sample volume of 1 mL and the sample was then kept at 37deg.C for 30 minutes. The pH was maintained at 8.5 with 0.3 M Tris Cl. Acrylamide, SDS, and Tris were eliminated by two cycles of centrifugation and dilution with distilled water in the centrifree ultrafiltration unit. Ten ul of sample were then spotted onto a Porton fiberglass peptide disk for sequencing. Note that the area of the Cys peak observed after cycle 4 is comparable to that of the Gln and Lys peaks observed on cycles 3, 2, and 6, indicating that the reaction of acrylamide with Cys was nearly quantitative.
Earlier work by Friedman and coworkers (1,2) indicates that the rate of reaction of acrylamide with Cys is similar to that of 4-vinyl pyridine. Thus most of the standard methods for alkylating Cys with 4-vinyl pyridine should work equally well when acrylamide is substituted for 4-vinyl pyridine. Experiments using 6 M guanidinium chloride in place of SDS to denature chymotrypsinogen (the first residue of which is Cys) and using 2 M acrylamide in place of 4-vinyl pyridine in the procedure described by Wilson and Yuan (3) also gave high yields of S-beta-propionamido Cys, supporting this conclusion (data not shown). A more detailed report on alkylating Cys with acrylamide for protein sequencing purposes will be presented elsewhere.
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