Estimation of Peptides

It is clear that the estimation of the amount of a particular peptide in a partial hydrolyzate may in certain cases yield considerably more information concerning the structure of the protein than its mere identification. Unfortunately it is impossible to determine directly the total amount of a particular peptide sequence in a protein molecule. Only a minimal value can be obtained from the composition of a partial hydrolyzate. In the case of the A-terminal peptides of insulin it was possible to estimate certain sequences in the protein from the yields of the peptides by allowing for the rate of breakdown of the bonds involved (p. 52). Such an approach may clearly be useful in the future where the yield of a peptide can be determined. 

Usually only small amounts of peptide are available after chromatographic fractionation of a partial hydrolyzate, so that micro methods of estimation are required. Consden et al. (1949) obtained an approximate estimate of peptides from a paper chromatogram by the color intensities of the amino acid spots produced on hydrolysis. Clearly careful control analyses with synthetic substances should accompany any estimaitons by this type of technique to allow for losses during fractionation. 

The use of isotopic methods for the estimation of peptides appears to offer considerable advantages of accuracy and general applicability. In the original isotope dilution technique of Rittenberg and Foster (1940) it was necessary to isolate a pure sample of the compound to be estimated, from a mixture of comparable amounts of other similar substances. In the case of peptides this would clearly be a formidable task, and has never been attempted. 

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For this reason either of these methods is suitable only for a comparative estimation of peptide content in urine. 

Figure 17.7 Hydrogen exchange kinetics of free ACTR and CBP and their complex. Representative spectra (a) and uptake curves (b) from peptides in CBP and ACTR that become a-helical in the complex. Secondary structure elements shown In diagram indicate a-helical (boxes), loop (lines), and unstructured (dotted lines) that ACTR adopts in complex with CBP [29]. The vertical dashed lines in the spectra denote the centroids of the undeuterated and fully deuterated states. The dashed lines in the uptake curves denote data fitting using Equation 17.20. (c) The kinetic analysis provides estimates of peptide-averaged protection, depicted as different colored bars mapped onto the sequence and secondary structure found in the complex. Adapted from Ref [81] with permission, 2011 American Chemical Society. (See insert for color representation of the figure.)...

Figure 5 Molecular mass estimation of peptide synthetases in one 3% SDS-PAGE. The molecular masses of the reference proteins recombinant tyrocidine synthetase 2 (190 kDa). myosin (205 kDa), and ACV synthetase (420 kD) are indicated. Extrapolation results in molecular masses of 570 kDa for gramicidin synthetase 2 (lane 1), 310,730, and 870 kDa for bacitracin synthetases 2, 1, and 3 (lane 2), 640 and 880 kDa for linear gramicidin synthetase 2 and tyrocidine synthetase 3 (lane 3), 1530 kDa for cyclosfwrin synthetase (lane 4), and 1460 kDa for peptolide SDZ 214-103 synthetase (lane 5). (From Ref. 58, with permission.)...

FIGURE 5.5 (a) The hydroxy amino acids serine and threonine are slowly destroyed during the course of protein hydrolysis for amino acid composition analysis. Extrapolation of the data back to time zero allows an accurate estimation of the amonnt of these amino acids originally present in the protein sample, (b) Peptide bonds involving hydrophobic amino acid residues snch as valine and isolencine resist hydrolysis by HCl. With time, these amino acids are released and their free concentrations approach a limiting value that can be approximated with reliability. 

Typically, the insertion induces sharp variation of the membrane profile at the distances 0.5-1.0nm from the membrane-peptide interface [79-82]. The steepness of this perturbation indicates that the short-A, behavior of membrane moduli must be important in the estimates of the elastic energy. In addition, a peptide inserted in a membrane almost certainly perturbs the membrane s elastic moduli in the immediate vicinity of the inclusion. Both these effects, membrane nonlocality and nonuniform modification of elastic properties by insertions, might play an important role in resolving the contradiction between the local calculations [80] and the experimental data for the mean lifetime of a gramicidin channel [81,109,110]. ... 

PJ Sinko, GD Leesman, GL Amidon. Mass balance approaches for estimating the intestinal absorption and metabolism of peptides and analogues Theoretical development and applications. Pharm Res 10 271-275, 1993. 

Fig. 37), suggesting that desolvation of the polar bonds in the molecule is a major determinant of permeability. Consistent with this, good correlations were found between the permeabilities of these peptides and their partition coefficients between heptane-ethylene glycol (r2 = 0.87) or the differences in partition coefficients between n-octanol-buffer and isooctane-buffer (r2 = 0.82) both these buffer systems provide experimental estimates of hydrogen-bonding potential. These results are qualitatively identical with those described earlier for the permeability of these peptides across Caco-2 cell monolayers. 

Estimated PPII Helix-Forming Propensities and Average Sum of Backbone ASAs from Monte Carlo Computer Simulations of Peptides Ac-Ala-Xaa-Ala-NMe Restricted to the PPII Conformation11... 

Fig. 2.5. Possible applications of a coupling parameter, A, in free energy calculations, (a) and (b) correspond, respectively, to simple and coupled modifications of torsional degrees of freedom, involved in the study of conformational equilibria (c) represents an intramolecular, end-to-end reaction coordinate that may be used, for instance, to model the folding of a short peptide (d) symbolizes the alteration of selected nonbonded interactions to estimate relative free energies, in the spirit of site-directed mutagenesis experiments (e) is a simple distance separating chemical species that can be employed in potential of mean force (PMF) calculations and (f) corresponds to the annihilation of selected nonbonded interactions for the estimation of e.g., free energies of solvation. In the examples (a), (b), and (e), the coupling parameter, A, is not independent of the Cartesian coordinates, x. Appropriate metric tensor correction should be considered through a relevant transformation into generalized coordinates...

A. Keller, et al., Empirical Statistical Model to Estimate the Accuracy of Peptide Identifications made by MS/MS and Database Search. Anal. Chem., 74, no. 1 (2002) 5383-5392. 

The simplest methods are usually restricted to the estimation of the amount of combined amino acids as a whole or of some definite fraction thereof separated from urine in certain fixed conditions. More efficient separation procedures permit identification of some simple peptides, which represent in many cases the nonphysiological constituents of abnormal urine. 

The determination of amino nitrogen before and after acid hydrolysis of urine has frequently been used for the quantitative estimation of the amount of urinary peptides (H5, M4). The number of liberated a-amino groups represents, in fact, the whole of formerly combined amino groups, not necessarily attached to a second amino acid partner. Besides, considerable losses connected with decomposition of some amino acids occur in the course of hydrolysis thus limiting the true quantitative value of this procedure. 

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