Amino acid analysis, protein characterization

The use of amino acid analysis to characterize protein hydrolyzates is well known, and IEC is an integral part of the commercial amino acid analyzer.61 Determinabon of individual amino acids in physiological fluids such as plasma and cerebrospinal fluid can be important in the etiology of various diseases.62 63 Of particular current interest is the measurement of total... 

Biomolecule Separations. Advances in chemical separation techniques such as capillary zone electrophoresis (cze) and sedimentation field flow fractionation (sfff) allow for the isolation of nanogram quantities of amino acids and proteins, as weU as the characterization of large biomolecules (63—68) (see Biopolymers, analytical techniques). The two aforementioned techniques, as weU as chromatography and centrifugation, ate all based upon the differential migration of materials. Trends in the area of separations are toward the manipulation of smaller sample volumes, more rapid purification and analysis of materials, higher resolution of complex mixtures, milder conditions, and higher recovery (69). 

Characterization of Purified Proteins Assessing purity, 182, 555 determining size, molecular weight, and presence of subunits, 182, 566 amino acid analysis, 182, 587 limited N-terminal sequence analysis, 182, 602 peptide mapping, 182, 613 analysis for protein modifications and nonprotein cofactors, 182, 626 protein crystallization, 182, 646. 

Two Streptomyces strains, S. badius and S. viridosporus, were found to be able to grow on kraft lignin (In-dulin ATR) as sole carbon source. The resulting APPL (Acid Precipitable Polymeric Lignin) was characterized by FTIR and elemental analysis for C, H and N, and was found to contain proteins in addition to a relatively demethoxylated lignin component. The proteins were further characterized by amino acid analysis, while the lignin component was separated by solvent extraction and its molecular weight distribution determined by HPSEC. 

Among the analytical methods presently used for the characterization of natural and synthetic peptides and proteins, the primary value of amino acid analysis is the determination of absolute peptide and protein content in solids and solutions and the quantitation of their amino acid composition and stoichiometry. It involves two steps, i.e. complete hydrolysis of peptides and proteins, followed by photometric determination of the released amino adds. The steps are laborious and time-consuming, and there is a continuous need for improvement of the techniques to increase precision and sensitivity. 

I n the previous three chapters we described the structures of amino acids and proteins, and in two cases we examined how these structures relate to their function. Some of the methods for structure determination were also discussed (e.g., sequence analysis in chapter 3 and x-ray diffraction in chapter 4). To analyze the structure of a protein we must isolate it from the complex mixture in which it exists in whole cells. The primary object of this chapter is to acquaint you with techniques used for protein purification. Because these procedures are often used for protein characterization as well, they will add to your repertoire of methods for protein characterization. 

During detailed characterization of a tryptic map of recombinant human tissue plasminogen activator (rtPA), a minor peak was resolved whose mass was not consistent with the expected set of tryptic peptides (L. Keyt and S.-L. Wu, unpublished observation). N-terminal sequence analysis of this fraction showed that it had a sequence containing residues 276-296 of rtPA with Lys-277 present as Hyl. We developed a modified amino acid analysis program capable of detecting hydroxylysine at levels down to 0.05 residues/mol to determine the distribution of Hyl in rtPA as well as in some other proteins derived from mammalian cells. 

Heterocyclic aromatic amines (HAA) are carcinogenic compounds that may occur in food. They are probably formed during cooking processes by the pyrolysis of amino acids and proteins. Since the mid 1990s, LC-MS plays a role in the analysis and characterization of these HAA. Both ESI-MS and APCl-MS are applied. A recent special issne of Journal of Chromatography B [82] emphasizes the analytical challenges related to HAA. The stractnres of the most abundant HAA in cooked food are shown in Figure 14.10. 

Amino acid analysis (AAA) is a classic analytical technique that characterizes proteins and peptides based on the composition of their constituent amino acids. It provides qualitative identification and is essential for the accurate quantification of proteinaceous materials. 

Note that analytical activities for protein pharmaceutical products follow similar principles but use methods for in vitro or in vivo (animal model) bioactivity, electrophoretic and chromatographic methods, amino acid analysis, peptide mapping, sequencing, etc., for characterization. 

The ideal candidate. .. will have a bachelor/masters degree in (bio)chemistry with 3 to 5 years experience in protein sequencing, and protein characterization using HPLC, mass spectrometry and amino acid analysis. 

Proteins and Amino Acids Total protein in food and feed samples is commonly determined by Kjeldahl (acid digestion/titration) or Dumas (pyrolysis) or elemental analysis.14 FIPLC can separate major proteins and furnish protein profiles and speciation information. HPLC can be used to further characterize specific proteins via peptide mapping and amino acid sequence analysis. HPLC modes used for protein include IEC, SEC, RPC, and affinity chromatography with typical UV detection at 215 nm or MS analysis. Details on protein separations are discussed in the life sciences section. 

Edman degradation was originally developed for determination of the primary structure (i.e., amino acid sequence) of peptides and proteins. Sequence analysis is not regularly performed for quality control in routine peptide synthesis but is more often employed for problem solving. As described earlier in this chapter, efficient characterization of synthetic peptides can be readily obtained by a combination of RP-EDPLC and mass spectrometry. Amino acid analysis is also valuable if MS is not available. If an incorrect mass or a discrepancy in the amino acid composition is found, one obvious alternative is to resynthesize the peptide. But, in order to deduce the cause of a failed synthesis, additional analyses must be performed. Both Edman degradation and tandem MS can be used to obtain sequence information... 

Determination of the amount and distribution of amino acids is required for a variety of bioanalytical questions. In bioprocess monitoring, the amount of certain free amino acids has to be monitored in order to avoid media depletion. In the case of a protein or a protein hydrolysate, analysis or verification of the amino acid composition may be a first step toward identification or characterization. The early work on amino acid analysis has been dominated by Stein and Moore, who in 1972 were awarded the Nobel prize... 

Thiazolidine residues have also been introduced into protein in other ways. Treatment of insulin in aqueous solution with the hydroxy-succinimide ester of 3-formyl-2,2-dimethyl-L-thiazolidine-4-carboxylic acid (353) yields three products, separable by chromatography. They are characterized, by end-group analysis, electrophoresis, and amino-acid analysis of the performic acid-oxidized chains, as monosubstituted derivatives (354) formed by interaction at each of the free amino-groups of... 

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