Proteins (polypeptides) are naturally occurring compounds that are composed of long chains of amino acids. Proteins are found throughout living things and function as hormones, structural elements, enzymes, immunoglobulins, and other constituents of living things. Research regarding the structure and functions of proteins often requires that the amino acid sequence (primary structure) of the protein be determined. In order for a protein or parts of a protein such as somatostatin, insulin, endorphins, etc. to be synthesized the sequence of amino acids must be determined before a synthesis can be attempted. In the search involving the function of proteins such as immunoglobulins, enzymes, viral coat proteins, and cell-surface proteins, the primary structure of the polypeptide must be determined in an attempt to elucidate the mechanism action of the protein.
The primary sequence of amino acids in proteins or polypeptides is commonly determined by stepwise chemical degradation process in which single amino acids are removed one by one from the end of the polypeptide for identification. The Edman degradation is the preferred method, while other methods have been developed and can be used in certain instances. In the Edman degradation amino acid removal from the end of the protein is accomplished by reacting the N-terminal amino acid residue with a reagent which allows selective removal of that residue from the protein. The resulting amino acid derivative is converted into a stable compound which can be chemically removed from the reaction mixture and identified.
Many physiologically active proteins are present in organisms at such extremely small concentrations that only very small amounts of the proteins can be obtained for sequencing analysis. Most current chemical sequencing methods are done with an amount of protein in the 5-100 nanomole (5.times.10.sup.-9 to 10.sup.-8 mole) range. It has been reported that micro sequencing of polypeptides by reverse phase high pressure liquid chromatography using ultraviolet light detection means has been accomplished with protein samples in the range of 50-500 picomole (5.times.10.sup.-11 to 5.times.10.sup.-10 mole) range. Other methods used in the micro sequencing of polypeptides involve radio labeling of the peptide or reagent, intrinsic radio labeling of the polypeptide, and enhanced UV detection of sequence degradation products, and others. While we will not discuss the details of these methods, each method has its limitations and restrictions. They have not been used with overall satisfactory results. The current best art is computer-aided UV detection of PTH amino acids from a microsequencer.
Many techniques have been developed in recent years for the analytical separation of polypeptides or proteins having high physiological activity in very low quantities of about 10.sup.-12 mole (1 picomole), about 50 nanograms of a 50,000 molecular weight protein. A sequencing method developed specifically for such small amounts of proteins is desirable.
One detection method which has an inherent sensitivity in the femtomole (10.sup.-13 to 10.sup.-15 mole) range is fluorescence. Attempts have been made to use fluorescent isothiocyanates in the Edman degradation, however overall success in micro sequencing of small amounts of protein has not been demonstrated to date.
Accordingly a substantial need exists for a micro sequencing reagent and method adapted for the sequencing of amounts of protein in the low picomole to sub-picomole range of protein.