Peptides spectrophotometric methods

Picric acid Suitable for extraction of amino acids and small peptides. Interferes with Kjeldahl or spectrophotometric methods Fox (1989), Salji and Kroger (1981), and Reville and Fox (1978)... 

Following successful recovery of peptide/protein molecule from the microspheres, a simple spectrophotometric method does not always allow discrimination between the monomeric protein form and its aggregates. However, HPLC might separate these species and thus provides more accurate qualitative data [96], But HPLC cannot quantify exclusively the amount of active protein antigen, as is the case with ELISA techniques [97], Nowadays, Fourier transform infrared (FTIR) spectroscopy has become a popular, noninvasive method, as it is able to characterize the secondary structure of entrapped proteins [26, 95, 98-101], Only recently, the integrity of their primary structure was evaluated, thanks to a new matrix-assisted laser... 

Peptide bond method. This spectrophotometric assay is based on the fact that peptide bonds absorb light in the 195 to 225 nanometer region of the spectrum. 

The identification of reactive or chemically modified residues of proteins is often extremely important for the characterization of proteins and their activity. Peptide mapping in conjunction with Edman sequencing and/or mass spectrophotometric analysis has been the method of choice to accomplish this characterization. However, this approach alone may not be sufficient or optimal for every situation as was the case when trying to identify the affinity ligand attachment sites on the B-chain of blocked ricin (Lambert et al., 1991a). 

Column separation of proteins and peptides is used for both preparative and analytical purposes. Generally, in the former case low- or medium-pressure systems are preferred, whereas in the latter, high-performance-liquid-chromatography (HPLC) is the method of choice. These systems are coupled with a spectrophotometric detector normally set at wavelengths 280 and (around) 220 nm for proteins and peptides, respectively. Coupling of HPLC with mass spectrometry allows structural identification of compounds after the separation. 

One practical alternative to the radiometric methods is provided by murexide, which forms a complex with free calcium (46). This complex can be determined spectrophotometrically and used for the calculation of unbound calcium. Bound calcium calculated by difference, enables one to construct binding plots. This method has two advantages in special cases it is very rapid and therefore can be used for labile binding proteins, and since no dialysis or ultrafiltration is needed it can be used with low molecular weight peptides. About 1 mg of protein is required for the murexide method. 

Several proteins can be cited (see Table VII) in which the agreement between the spectrophotometric figures for tyrosine and tryptophan and the analytical data obtained by other methods is sufficiently close to justify the conclusion that in these proteins there cannot be any significant contribution to the absorption spectrum by the peptide fabric, in the 2700-3100 A. region. Any such contribution would have seriously affected the accuracy of the spectrophotometric analysis. 

Biuret method Peptide bonds in a enzyme react with the Cu(ll) ion in a strongly alkaline solution and produce a complex that can be determined spectrophotometrically at 540 or 310nm 0.2-10 mg ml" (540 nm) 30-500ngml" (310nm)... 

Stability of ttT tophan to anhydrous HF (Sakaldbara et al., 1967) was confirmed when two tryptophanyl peptides, Try-Phe and Try-Leu-Met-Asn-Thr, were synthesized by the solid-phase method and cleaved both by HF and HBr/TFA. In both cases the material resulting from the HF cleavage appeared to have less tryptophan degradation as evidenced spectrophotometrically and by TLC. 

A simple experiment in which these parameters can be obtained by conventional experimental methods uses papain as the enzyme and Azocoll as the substrate. Papain is a sulph-hydryl protease that can be isolated from the fruit papaya (paw-paw), and which is used for tenderising meat. Azocoll is insoluble ground cowhide to which a red dye has been covalently bonded. When the peptide bonds of the collagen are hydrolysed by the papain, the dye is liberated into the solntion and the increase in its concentration as a function of time can be studied spectrophotometrically at 520 nm. 

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