A new approach for the solid phase sequence determination of proteins

Abstract

The use of the modified Edman reagent, 4-N,Ndimethylaminoazobenzene 4’-isothiocyanate, in the stepwise degradation of peptides and proteins has provided a useful method for sequence determination [l-3] . Released N-terminal amino acids are identified as 4-N,N-dimethylaminoazobenzene 4’-thiohydantoins directly by thin-layer chromatography (t.1.c.) on polyamide sheets, the intense colour of the amino acid derivatives allowing detection in subnanomol quantities. Low repetitive yields due to extraction losses and time-consuming operations at each drying step have reduced both sensitivity and efficiency of the manual sequencing work. However, the solid phase degradation of Laursen [4,5] whereby peptides are attached covalently to an insoluble support, eliminates extraction losses and shortens greatly the time needed for the drying of residues after the extractions. A solid phase degradation procedure using 4-N,Ndimethylaminoazobenzene 4’-isothiocyanate has been developed. The N-terminal sequence of native lysozyme (first 19 residues) and glucagon (first 10 residues) attached to controlled porosity glass beads activated by p-phenylene diisothiocyanate have been obtained. Reduced Histidinol dehydrogenase (mol. wt 40 000) from Salmonella typhimwium, of hitherto unknown sequence has been attached via cysteine residues to an iodoacetamide derivative of controlled porosity glass beads and the first eight amino acid residues determined. This new solid phase sequenching method is sensitive and efficient, requiring practically, 210 nmol protein as the starting material. Each cycle of