Determination of Primary Structure

Determine the amino acid composition and molecular weight of the pure protein. From amino acid composition and molecular weight data, calculate the number of residues of each amino acid present per protein molecule to the nearest whole number. 

Determine amino terminal (N-terminal) and carboxy terminal (C-tcrminal) amino acids. (A unique residue for each terminus suggests that the native protein contains only one peptide chain.) 

Fragment aliquots of the polypeptide by enzymatic or chemical hydrolysis and separate the peptide mixtures into individual fragments. (This process will yield overlapping sets of smaller peptides.) 

Assume that cleavage of the above polypeptide with a specific cleaving agent yields four peptides. A, B, C, and 

These peptides are separated and sequeneed but it is yet not known whether the correct order is ABCD or ACBD. A second hydrolytic procedure gives rise to three peptides  

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MOLECULAR BIOLOGY HAS REVOLUTIONIZED THE DETERMINATION OF PRIMARY STRUCTURE... 

In recent years, C-NMR spectroscopy has found extensive use in studies on carbohydrate polymers, in some series overshadowing the importance of H-NMR spectroscopy. Applications range through determination of primary structure, analysis of mixtures, monitoring of chemical and enzymic transformations or of chelation reactions, and studies on conformational change. Measurements of coupling are utilized in determining the... 

Table I. Techniques Used for Determination of Primary Structure...

The fundamental work of Lemieux and coworkers21 introduced the successful application of H-n.m.r. spectroscopy to structural problems in the carbohydrate field. Since then, numerous studies have been devoted to the (partial) determination of primary structures of carbohydrates and derivatives thereof, as well as to the elucidation of their conformation in solution, by means of H-n.m.r. spectroscopy. An exhaustive discussion of all these contributions is beyond the scope of this article. For comprehensive reviews, see Refs. 22-28. 

Lee, R. C., Burton, R. A., Hrmova, M., and Fincher, G. B., Barley arabinoxylan arabinofuranohydrolases purification, characterization and determination of primary structures from cDNA clones. Biochem 72001, 356 (Pt 1), 181-9. 

Several physicochemical methods of investigation, such as measurement of optical rotation (including optical rotation dispersion and circular dichroism) rheological methods, and X-ray analysis of fibers and films, have played a very important role for determination of conformations of galactan molecules and characterization of their supramolecular structures.8,19 As mentioned in Section I, only physicochemical methods used for determination of primary structures of polysaccharides are considered in this chapter. 

Hrmova M, Harvey AJ, Wang J, Shirley NJ, Jones GP, Stone BA, Hoj PB, Fincher GB (1996) Barley /3-D-Glucan Exohydrolase with /J-D-Glucosidase Activity. Purification, Characterization, and Determination of Primary Structure from a cDNA Clone. J Biol Chem 271 5277... 

The first step in determination of primary structure is hydrolysis and quantitative analysis of amino acid composition by ion-exchange chromatography. 

DETERMINATION OF PRIMARY STRUCTURE AMINO ACID SEQUENCING... 

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