Absorption peptide bond

Proteases are enzymes that break peptide bonds in proteins. As such they lend themselves to a variety of homogeneous assay techniques. Most employ labeling both ends of the substrate with a different tag, and looking for the appearance (disappearance) of the signal generated in the intact substrate (product). As an example, for a fluorescence quench assay, the N-terminal of a peptide is labeled with DNP and the C-terminal with MCA. As such, the peptide is fluorescently silent since the fluorescence from DNP is quenched by absorption by the MCA. Another very popular donor/acceptor pair is EDANS 5-[(2-aminoethyl)amino] naphthalene-1-sulfonic acid and DABCYL 4-(4-dimethylaminophenylazo)benzoic acid) (a sulfonyl derivative (DABSYL) [27], Upon peptide cleavage, the two products diffuse, and due to a lack of proximity, the fluorescence increases. 

A careful comparative pharmacokinetic study of the tripeptoid 6.108 and the tetrapeptide N-phenylacetyl-Leu-Asp-Phe-D-Pro-amide (6.109) in rats has provided insights on absorption and disposition [233]. The two compounds have comparable backbone structures but differ in the presence or absence of peptide bonds. They also have similar octanol/water partition coefficients, although the H-bonding capacity of the tetrapeptide is greater. In an in vitro model, the two compounds had comparable, and low, absorption clearances (6.7 x 10 4 vs. 4.8 x 10 4 ml min-1 cnT1 for the peptoid and the... 

These proteolytic enzymes are all endopeptidases, which hydrolyse links in the middle of polypeptide chains. The products of the action of these proteolytic enzymes are a series of peptides of various sizes. These are degraded further by the action of several peptidases (exopeptidases) that remove terminal amino acids. Carboxypeptidases hydrolyse amino acids sequentially from the carboxyl end of peptides. They are secreted by the pancreas in proenzyme form and are each activated by the hydrolysis of one peptide bond, catalysed by trypsin. Aminopeptidases, which are secreted by the absorptive cells of the small intestine, hydrolyse amino acids sequentially from the amino end of peptides. In addition, dipeptidases, which are structurally associated with the glycocalyx of the entero-cytes, hydrolyse dipeptides into their component amino acids. 

In D O solution, the cyclic trimeric derivative of azetidine-2-carboxylic acid [cyclo(Aze)3] displayed (78MI2) more than one interconverting conformation, with peptide bonds slightly deviated from planarity. Circular di-chroism in methanol showed (78MI3) absorption very similar to that of... 

Cheeseman et al. reported in 1972 the purification and properties of a fluorescent compound from Methanobacterium. Owing to the first visible absorption at 420 nm the unknown compound was called Factor-420. It was not until 1978 till it became clear that the compound is a flavin-like molecule By different physical techniques it could be shown that the molecule is a 5-deaza-FMN derivative (Scheme 2, (70)) where at position 8 the methyl group is replaced by a hydroxy group. In addition, the side chain phosphate group is esterified by a lactyl group which, in turn, is linked to a diglutamyl moiety via a peptide bond. Factor-420 functions as... 

Accordingly, the quantitation of proteins by peptide bond absorption at 205 nm (A2os) is more universally applicable among proteins. Furthermore, the absorptivity for a given protein at 205 nm is several-fold greater than that at 280 nm (Scopes, 1974 Stoscheck, 1990). Thus lower concentrations of protein can be quantitated with the A205 method. The disadvantage of this method is that some buffers and other components absorb at 205 nm (Stoscheck, 1990). 

Absorbance of peptide bonds at 2170 nm is usually used as a key band for the calibration of protein content measurement. However, the intensity of the absorption depends on the structure and conformation of the protein. Yamashita et al [8] investigated the change in absorption at 2170 nm caused by the conformational change of protein using bovine serum albumin (BSA) as the model protein. A mixture of dithiothreitol (a reducing agent) and BSA (5%) was taken in a quartz cell attached to the NIR instrument, and then spectra were recorded at 10-minute intervals. Dithiothreitol reduced the disulfide bond in BSA. The... 

C. Determination of Protein Concentration by Peptide-Bond Absorptivity. 380... 

The ultraviolet spectra of proteins can for present purposes be divided into three regions above 2500 A, between 2500 and 2100 A, and below 2100 A. We can paraphrase this division by calling the first region simple —only a few absorbers and easily sorted out calling the second region complex because of the multiplicity of the contributions to absorptivity and calling the third complex but exciting, because of the peptide-bond absorption and its conformation-dependence. 

Two factors have led to recent activity in studying the shorter-wave region. First, the means several commercial spectrophotometers of high quality, capable of operating to appreciably below 2000 A, have become available in the past few years. Second, the objective the attraction of studying the absorptivity of that most characteristic component of protein structure, the peptide bond itself. Because of its fundamental importance for all protein studies, this aspect of protein spectra will be emphasized in the present discussion. 

Exciton theory deals with the theory of electronic excitation processes in ordered arrays of absorbers (Kasha, 1959). Applications of exciton theory to protein problems have not been numerous, but some important results, especially on the conformation-dependence of the peptide absorption around 2000 A, will be discussed in the section on peptide-bond absorption. 

Solid-state spectral studies relating to proteins have not been numerous, but those associated with the peptide-bond absorption are of special interest. In particular, we note Peterson and Simpson s (1957) singlecrystal spectroscopy of myristamide, and the demonstration of dichroism in oriented films of helical polypeptides by Gratzer et al. (1961). The results of these studies are discussed in Section IV, . Since great care and exacting technique is required for solid-state studies, we may do no better than to refer interested readers to the original papers for critical discussions of experimental aspects. 

Of the various arguments and evidence presented by Glazer and Smith, the following offer the strongest support, in the writer s opinion, for the possibility of absorptivity contributions in the 2300 A region from peptide bonds. 

We may also mention that peptide-bond absorption at somewhat longer wavelengths has been employed by Mitz and Schleuter (1957), and by Schmitt and Seibert (1961) to estimate the rates of digestion by peptidases acting on simple peptides. Schwert and Takenaka (1955) describe difference-spectral methods for assaying chymotrypsin and trypsin with acetyl-L-tyrosine ethyl ester and benzoyl-L-arginine ethyl ester as the respective substrates. 

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