Peptide examination

In 1961 Ansorge et al. (A5), using the same technique as previously, determined the peptide composition of urine derived from four normal subjects, three males and one female. Among twenty isolated peptides, seventeen were found in all specimens of urine, two peptides in three specimens, and the remaining one only in two specimens of urine. The identity of individual peptides was established on the basis of their electrophoretic and chromatographic behavior, as well as the amino acid composition after complete hydrolysis. It should be pointed out, however, that the amino acid composition of the peptides examined differs considerably from that obtained by the same authors in the case of the peptides described in 1958 (HI). 

In Sect. 6.1, we defined a peptide as any compound produced by amide formation between a carboxy group of one amino acid and an amino group of another. Our definition is explicit as far as the relation between component residues is concerned, but is incomplete since the definition of amino acids is left open. In Sect. 6.3 and 6.4, we used a narrow definition of amino acids (i.e., proteinogenic a-amino acids). In contrast, the peptides examined... 

Of the peptides examined, none contained arginine. Bonds formed by this amino acid should be very susceptible to hydrolysis. 

Stopped-flow fluorescence studies of ES complexes provided a direct comparison of the peptide binding aflBnities of the zinc and cadmium enzymes and, simultaneously, an explanation for the different roles of metals in peptide and ester hydrolysis (48). Cadmium carboxypeptidase binds the peptide Dns-(Gly)3-L-Phe as readily as does [(CPD)Zn] but catalyzes its hydrolysis at a rate that is reduced considerably (Figure 8). Initial rate studies of oligopeptides are in agreement with this observation. For all peptides examined, the catalytic rate constants of the cadmium enzyme are decreased markedly, but the association constants (1) (Km values) of the cadmium enzyme are identical to those of the zinc enzyme (48,51,57). However, in marked contrast, for all esters examined the catalytic rate constants of the cadmium enzyme are nearly the same as those of the zinc enzyme, but the association constants are decreased greatly. 

Some peptides bearing a basic moiety in the side chain of the amino acid are also candidates as a catalyst for cyanation [20,21,22,23]. Among the acyclic as well as the cyclic peptides examined, a cyclic dipeptide (5 Fig. 1) composed of (S)-phenylalanine (Phe) and (S)-histidine (His) was found to be effective [24,25,... 

The primary structure of a peptide is its ammo acid sequence We also speak of the secondary structure of a peptide that is the conformational relationship of nearest neighbor ammo acids with respect to each other On the basis of X ray crystallographic studies and careful examination of molecular models Linus Pauling and Robert B Corey of the California Institute of Technology showed that certain peptide conformations were more stable than others Two arrangements the a helix and the (5 sheet, stand out as... 

For nonvolatile or thermally labile samples, a solution of the substance to be examined is applied to the emitter electrode by means of a microsyringe outside the ion source. After evaporation of the solvent, the emitter is put into the ion source and the ionizing voltage is applied. By this means, thermally labile substances, such as peptides, sugars, nucleosides, and so on, can be examined easily and provide excellent molecular mass information. Although still FI, this last ionization is referred to specifically as field desorption (FD). A comparison of FI and FD spectra of D-glucose is shown in Figure 5.6. 

Although simple solutions can be examined by these electrospray techniques, often for a single substance dissolved in a solvent, straightforward evaporation of the solvent outside the mass spectrometer with separate insertion of the sample is sufficient. This situation is not true for all substances. Peptides, proteins, nucleotides, sugars, carbohydrates, mass organometallics, and many... 

For mixture.s the picture is different. Unless the mixture is to be examined by MS/MS methods, usually it will be necessary to separate it into its individual components. This separation is most often done by gas or liquid chromatography. In the latter, small quantities of emerging mixture components dissolved in elution solvent would be laborious to deal with if each component had to be first isolated by evaporation of solvent before its introduction into the mass spectrometer. In such circumstances, the direct introduction, removal of solvent, and ionization provided by electrospray is a boon and puts LC/MS on a level with GC/MS for mixture analysis. Further, GC is normally concerned with volatile, relatively low-molecular-weight compounds and is of little or no use for the many polar, water soluble, high-molecular-mass substances such as the peptides, proteins, carbohydrates, nucleotides, and similar substances found in biological systems. LC/MS with an electrospray interface is frequently used in biochemical research and medical analysis. 

Evaporation from a spray of charged droplets produced from a stream of liquid yields ions that can be analyzed in a mass spectrometer. Thermally labile and normally nonvolatile substances such as sugars, peptides, and proteins can be examined successfully. 

The importance of linked scanning of metastable ions or of ions formed by induced decomposition is discussed in this chapter and in Chapter 34. Briefly, linked scanning provides information on which ions give which others in a normal mass spectrum. With this sort of information, it becomes possible to examine a complex mixture of substances without prior separation of its components. It is possible to look highly specifically for trace components in mixtures under circumstances in which other techniques could not succeed. Finally, it is possible to gain information on the molecular structures of unknown compounds, as in peptide and protein sequencing (see Chapter 40). 

Intact peptides and proteins can be examined by a variety of new techniques, including MS/MS, dynamic FAB, APCI, and electrospray. Large masses of tens of thousands of Daltons can be accurately measured with unprecedented accuracy by electrospray. 

An alternative approach to peptide sequencing uses a dry method in which the whole sequence is obtained from a mass spectrum, thereby obviating the need for multiple reactions. Mass spec-trometrically, a chain of amino acids breaks down predominantly through cleavage of the amide bonds, similar to the result of chemical hydrolysis. From the mass spectrum, identification of the molecular ion, which gives the total molecular mass, followed by examination of the spectrum for characteristic fragment ions representing successive amino acid residues allows the sequence to be read off in the most favorable cases. 

Both FI and FD provide good molecular mass information, but few if any fragment ions, and allow thermally labile substances such as peptides, nucleosides, and glycerides to be examined, as well as inorganic salts. 

Elber et al. [48] applied this method to explore the dynamics of the C-peptide in water with impressive results. More than 30 trajectories of C-peptide were generated, and the process of helix fonnation in water was examined. Remarkably, a time step of 500 ps was used, which allowed for the study of peptide folding on extended time scales. 

In this chapter we shall illustrate some fundamental aspects of enzyme catalysis using as an example the serine proteinases, a group of enzymes that hydrolyze peptide bonds in proteins. We also examine how the transition state is stabilized in this particular case. 

In order to examine whether this sequence gave a fold similar to the template, the corresponding peptide was synthesized and its structure experimentally determined by NMR methods. The result is shown in Figure 17.15 and compared to the design target whose main chain conformation is identical to that of the Zif 268 template. The folds are remarkably similar even though there are some differences in the loop region between the two p strands. The core of the molecule, which comprises seven hydrophobic side chains, is well-ordered whereas the termini are disordered. The root mean square deviation of the main chain atoms are 2.0 A for residues 3 to 26 and 1.0 A for residues 8 to 26. 

The situation is different for other examples—for example, the peptide hormone glucagon and a small peptide, metallothionein, which binds seven cadmium or zinc atoms. Here large discrepancies were found between the structures determined by x-ray diffraction and NMR methods. The differences in the case of glucagon can be attributed to genuine conformational variability under different experimental conditions, whereas the disagreement in the metallothionein case was later shown to be due to an incorrectly determined x-ray structure. A re-examination of the x-ray data of metallothionein gave a structure very similar to that determined by NMR. 

This section discusses in detail the column types that are available for the size exclusion chromatography of both polar and nonpolar analytes. It first discusses the various columns available for standard nonaqueous size exclusion chromatography. It then reviews the columns available for general size exclusion chromatography using aqueous mobile phases. Finally, it examines the columns designed for size exclusion chromatography of proteins and peptides. 

The enzymatic cleavage of esters is a vast and extensively reviewed area of chemistry. Recently, several new esters have been examined primarily for the preparation of peptides and glycopeptides. 

Dioxo-2, 4, 5 -trimethylcyclohexa-l, 4 -diene)-3,3-dimetbylpropi-onamide (Q). The application of this well-known acid [3-(3, 6 -dioxo-2, 4, 5 -trimethylcyclohexa-l, 4 -diene)-3,3-dimethylpropionic acid] to protection of the amino function for peptide synthesis has been examined. Reduction of the quinone with sodium dithionite causes rapid trimethyl lock -facilitated ring closure with release of the amine. 

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