Enzymatic sequence analysis

Scheme 1. Wiebers s combined MS-enzymatic sequence analysis method. At the dinucleotide level one can already make an identification of the remaining sequence (FD). N stands for nucleoside.

PRIMARY STRUCTURE OF PROTEINS CHEMICAE AND ENZYMATIC SEQUENCE ANALYSIS 95... 

PRIMARY STRUCTURE OF PROTEINS CHEMICAL AND ENZYMATIC SEQUENCE ANALYSIS 97... 

Scharf, S.J., Horn, G.T. and Erlich, H.A. (1986) Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science 233,1076-1078. 

To facilitate sequencing analysis and enzymatic cleavage, reduction and alkylation of the peptides to open their potential disulfide bridges is recommended. Several alkyl groups can be used acrylamide, iodoacetamide, and 4-vinylpyridine (4-VP). S-pyridylethylation is preferred over the other alkyl groups when Edman degradation (PTH-4-pyridylethylated-Cys is commercially available) and MS (addition of 57 Da per alkylated cysteine residue) have to be performed. 

During the period of time when the nature of the 19-nortestosterone acetate-dependent photoinactivation was under investigation, a new bacterial steroid isomerase was obtained from extracts of Pseudomonas putida (Biotype B) in nearly homogeneous form and some of its physical and enzymatic properties were characterized (64, 65, 66). The putida isomerase is similar in its molecular weight and quaternary structure to the testosteroni isomerase. Chemically, the most striking difference between the two isomerases is the presence of four residues of cysteine per polypeptide chain of the putida isomerase whereas no cysteine or cystine is present in the testosteroni isomerase. N-Terminal sequence analysis of the putida isomerase demonstrated substantial sequence homology between the two enzymes. 

Fernandez, J., DeMott, M., Atherton, D., and Mische, S. (1992). Internal protein sequence analysis enzymatic digestion for less than 10 micrograms of protein bound to polyvinylidene difluoride or nitrocellulose membranes. Anal. Biochem. 201, 255-264. 

Jones, B. N., Paabo, S., and Stein, S. (1981). Amino acid analysis and enzymatic sequence determination of peptides by an improved o-phthaldialdehyde precolumn labeling procedure. J. Liq. Chro-matogr. 4, 565-586. 

Despite the limitations of partial acid hydrolysis it has been employed by several investigators for analysis of small peptides which were produced by enzymatic hydrolysis of large polypeptides or proteins (e.g., Harris and Roos, 1959a,b Margoliash, 1962 Dus et al., 1962). When used properly partial acid hydrolysis should remain a valuable technique in sequence analysis. 

Quantitative measurement of phospholipids is rare in routine clinical practice but more common in research (e.g., in studies of dietary influences). The choline-containing phospholipids lecithin, lysolecithin, and sphingomyelin, which account for at least 95% of total phospholipids in serum, are readily measured by an enzymatic reaction sequence using phospholipase D, choline-oxidase, and horseradish peroxidase. Kit methods with this enzymatic sequence are available commercially. Before the availability of enzymatic reagents, the common quantitative method involved extraction and acid digestion with analysis of the total lipid-bound phosphorus. ... 

Modification of the prcformulation format for biotechnological products from the original guidance must be considered. The sections regarding chemical structure, physicochemical properties, and stability may be revised according to the nature and characteristics of proteins and peptides. Aside from the conventional analytical instruments and techniques used in the study of small molecules, methods such as amino acid analysis, sequence analysis bioassay, immunoassay, and enzymatic assay are commonly used and should be included in the report. 

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