Proteins elemental analysis

The natural world is one of eomplex mixtures petroleum may eontain 10 -10 eomponents, while it has been estimated that there are at least 150 000 different proteins in the human body. The separation methods necessary to cope with complexity of this kind are based on chromatography and electrophoresis, and it could be said that separation has been the science of the 20th century (1, 2). Indeed, separation science spans the century almost exactly. In the early 1900s, organic and natural product chemistry was dominated by synthesis and by structure determination by degradation, chemical reactions and elemental analysis distillation, liquid extraction, and especially crystallization were the separation methods available to organic chemists. 

Subsequent studies provided a wealth of information that appeared to support the hypothesis that the Fepr protein was a genuine [6Fe-6S]-containing protein. In a biochemical study (10) the elemental analysis was meticulously repeated, and, based on an assumed molecular mass of 52 kDa, the prismane protein was found to contain 6.3 Fe atoms, averaged over as many as nine different preparations. Again, no other metals than Fe were detected, suggesting that all... 

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. 

One type of the constituent metallocenters in the MoFe protein has the properties of a somewhat independent structural entity. This component, referred to as the FeMo cofactor (FeMo-co), was first identified by Shah and Brill (1977) as the stable metallocluster extracted from acid-denatured MoFe protein. The FeMo-co was able to fully activate a defective protein in the extracts of mutant strain UW45, a protein which subsequently was shown to contain the P clusters but not the EPR-active center. The isolated cofactor accounted for the total S = t system observed by EPR and Mdssbauer spectroscopies of the holo-MoFe protein (Rawlings et al., 1978). Elemental analysis indicated a composition of Mo Fee-8 Se-g for the cofactor, which, if there are two FeMo-co s per a2 2> accounts for all the molybdenum and approximately half the iron in active enzyme (Nelson etai, 1983). Although FeMo-co has been extensively studied [reviewed in Burgess (1990)] the structure remains enigmatic. To date, all attempts to crystallize the cofactor have failed. This is possibly due to the instability and resultant heterogeneity of the cofactor when removed from the protein. Also, there is a paucity of appropriate models for spectral comparison (see Coucouvanis, 1991, for a recent discussion). Final resolution of this elusive structure may require its determination as a component of the holoprotein. 

Ldnnerdal, B., Stanislowski, A. G., Hurley, L. S. J. Inorg. Biochem. 12, 71 (1980) Woittierz, J. R. W. Elemental Analysis of Human Serum and Human Serum Protein Fractions by Thermal Neutron Activation, Netherlands Energy Research Foundation Report, ECN 147, January 1984... 

Figure 6. Multivariate factor analysis of beef showing the " distribution" of experimental treatments, volatile chemical attributes, chemical protein elements and flavor attributes in beef samples during postmortem aging. Insert represents the data redrawn with the graphical origin depicted as the fulcrum of a balance beam.

Table 9.28 Combination of element analysis using LA-ICP-MS and protein identification (Alzheimer disease) by MALDI-FTICR-MS.

The applications based on point (1) are the so-called speciation analysis dealing with organome-tallic compounds, proteins, elements in different oxidation states and similar analytical problems... 

Since the first purifications of Fe hydrogenases in the early 1970s a range of different models for the H-cluster active site have been proposed including mononuclear iron and clusters of 2, 3, 4, and 6Fe [56,57,72-77], At least in part this changing stoichiometry reflects improvements in purification, elemental analysis, and spectroscopy. The more recent models propose the H cluster to contain approximately 6 Fe on the basis of elemental analysis [56,57] and a putative 3.3 A Fe-Fe distance indicated by EXAFS spectroscopy [58], The data are indicative at best, because counting Fe in proteins has an uncertainty typically of the order of 10% (i.e., l-2Fe), and because no EXAFS on 6Fe models has been published. 

The complexity of quality control for proteins, as compared to small molecules, is most evident in the requirements for proof of structure. Many small molecules can be fully characterized using a few spectroscopic techniques (e.g., NMR, IR, mass spectrometry, and UV) in conjunction with an elemental analysis. However, proving the proper structure for a protein is much more complex because 1) the aforementioned spectroscopic techniques do not provide definitive structural data for proteins, and 2) protein structure includes not only molecular composition (primary structure) but additionally, secondary, tertiary, and, in some cases, quaternary features. Clearly, no single analytical test will address all of these structural aspects hence a large battery of tests is required. 

J. R. W. Woittiez, On the use of separation techniques for elemental analysis of protein fractions, in P. Bratter, B. Ribas, P. Schramel (eds), Trace Element Analytical Chemistry in Medicine and Biology, Vol. 6, Consejo Superior De Investigations Cientihcas, Madrid, 1994, pp. 1-32. 

The first step, as alluded to above, is the development of possible loop conformations which connect the regions of secondary structure The loops which do not fit into the well-defined category of a-helices or (1-sheets have been fairly well characterized using the data base of proteins for which the three-dimensional structure is known [15,16], The identification of specific loop conformations provides insight into the possible orientations, or at least provides limitations on the possible orientations, of the various secondary structural elements. The second step is then analysis of the array of amino acids within the secondary structural elements with attention to the environment in which the amino acids would be found. It is clear that a cluster of hydrophobic amino acids would not likely be projecting into the aqueous solution, and more likely projecting into the core of the protein. This analysis provides additional restrictions to the number of possible arrangements in which the secondary structural elements may be found. 

Although AOT is also an anionic surfactant of the same type as DOLPA, haemoglobin cannot be transferred into the AOT reverse micellar phase, and most haemoglobin can be seen at the oil-water interface as a red precipitate. Adachi and Harada have reported that cytochrome c precipitated as a cytochrome c-AOT complex at low concentrations of AOT [7]. It was found that this precipitate is likewise the AOT-haemoglobin complex (AOT/haemoglobin = 120 1) from the results of elemental analysis [8]. These results indicate that the difference in the extraction ability of DOLPA and AOT might depend on the hydrophobicity of the surfactants provided to the hydrophilic proteins. 

The most commonly submitted samples for direct trace element analysis are of whole blood, blood plasma, or serum. Plasma protein levels of the relevant carrier proteins transferrin (Fe), albumin (Zn), ceruloplasmin (Cu), and selenoprotein P (Se) can give useful additional information. 

In the past 10 years, a wide variety of resins have been used to fractionate humates. Interest in these resins originates from a search for methods to concentrate materials from natural waters. It is necessary to mention the first resin used to fractionate humates charcoal (Forsyth, 1947). Carbohydrates and proteins were preferentially separated by this method. Anderson and Russell (1976) purified fulvic acid by a charcoal separation. Elemental analysis showed this fulvic acid to be identical to a direct citric acid extract of the soil. 

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. 

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