Alex G. Bonner

Solid-phase Edman degradation. The use of p-phenyl diisothiocyanate to attach lysine- and arginine-containing peptides to insoluble resins

The primary step in sequencing peptides by the solid-phase Edman degradation [l] is attachment of the peptide to an insoluble resin, generally by blocking the peptide amino groups and then activating the C-terminal carboxyl with carbonyldiimidazole [l] or a carbodiimide [2-41. A problem limiting the usefulness of the solid-phase method is that the side-chain carboxyls of aspartic and glutamic acids also become activated, with the result, at least when carbonyldiimidazole is used, that the y-carboxyl of glutamic acid becomes bound to the resin and that aspartic acid apparently forms a cyclic imide which prevents further degradation of the peptide [i] . Although procedures for selective blocking of side chain carboxyls have been devised [2], they involve a number of steps and are often unsuitable for use with nanomole amounts of peptide. A procedure we have found to be particularly useful for tryptic peptides involves attaching them by their lysine e-amino groups to aminopolystyrene usingp-phenyl diisothiocyanate. Arginine peptides are first deguanidated with hydrazine [5],

The amino acid sequence of a dimeric myoglobin from the gastropod mollusc, Busycon canaliculatum L

Several years ago Read et al. [l-6] described the isolation and characterization of the radular muscle myoglobins from several gastropod and amphineuran molluscs. All of these proteins are dimeric, with molecular weights of about 34 000; those from Busycon canaliculatum and Buccinum undatum are particularly interesting in that they show cooperative binding of oxygen (Hill coefficients of n 2 1.1 and IZ = 1.4, respectively [l]). Since these proteins occupy a position between the tetrameric hemoglobins of vertebrates and the monomeric myoglobins, we felt it would be of value to investigate their primary structures as a first step towards understanding how they bind oxygen. The amino acid sequence of the Busycon myoglobin was determined and found to be homologous with, but about 80% different from, all other related globins of known sequence.

Sensitivity Enhancement in Solid-Phase Protein Sequence Analysis: An Examination of Methodology

One of the early impacts of the genetic engineering revolution has been a marked increase in interest in amino acid sequence analysis of proteins and peptides, especially on the so-called “micro” level. The scope of the term “microsequencing”, however, has yet to be defined. As such, the question of sensitivity in protein sequence analysis is continually being raised.

Solid Phase Synthesis of Polypeptides and Oligonucleotides Using a Solid Phase Sequencer

Recent experiments with a Sequemat solid-phase sequencer indicate the feasibility of adapting this instrument to perform the solid phase synthesis of polypeptides and oligonucleotides. Only programming and minor plumbing changes are necessary to reversibly convert the Sequemat sequencer into a solid phase synthesizer. Microbore columns were used with polystyrene, acrylamide and silica resins diluted with unfunctionalized glass beads to provide back pressures of 150–190 psig. The usual scale of the reactions provided 0.001 to 0.1 mmole of polypeptide or oligonucleotide. The cycle time was 1–1.5 hours per synthetic step with each step providing only 25–50 ml total waste volume.