Peptide and proteins

On the other hand, denaturation by such reagents results in loosening of the original protein structure and the polypeptide chain configuration becomes a random coil which leads to changes in retention volume. This fact has to be taken into account especially in molar mass determinations. SDS is bound to protein propor- 

In dogs with experimental coronary thrombosis samples of blood were taken from the aorta and coronary sinus at intervals of 2, 4 and 24 h and subjected to gel chromatography [80]. A deviation from the normal pattern was observed as early as after 2 h. Such an early change in the characteristics of the chromatographic elution curve is a prerequisite for GPC being used in early diagnosis with eventual application in man. 

Gibb et al. [81] compared the properties of normal sera from various fish species by sedimentation analysis and gel chromatography. By the latter, the sera were separated into three main fractions. The serum from each species gave a specific elution curve. 

Gel chromatography may prove useful for the separation of biomacromolecules at different degrees of polymerization, as is the case with synthetic polymers for example, Nakasaki et al. [89] purified alkaline phosphatase from rat small gut. One of the purification steps was gel chromatography on a porous glass column-filling material which resulted in separation of the enzymatic activity into three fractions. Further analysis of the fractions by polyacrylamide gel electrophoresis in the 

GPC proved of great value in the preparation of viral proteins, because the fractions obtained could be subjected to further analysis. A study of viral proteins is of interest from the point of view of not only the virion structure but also the immunological properties. 

Proteins and peptides are linear polymers made up of combinations of the 20 most common amino acids linked with each other by peptide bonds. Moreover, the protein produced by the ribosome may undergo covalent modifications, called post-translational modifications, after its incorporation of amino acids. Over 200 such modifications have been detected already [13,14], the most important being glycosylation, the formation of disulfide bridges, phosphorylation, sulfation, hydroxylation, carboxylation and acetylation of the N-terminal acid [15]. The most frequent are listed in Table 8.1 and a more comprehensive database of mass changes due to post-translational modifications of peptides and proteins is available on the Internet [16]. 

Mass spectrometry not only allows the precise determination of the molecular mass of peptides and proteins but also the determination of their sequences, especially when used with tandem mass techniques. Indeed, fragmentation of peptides and proteins gives sequence information that can be used for protein identification, de novo sequencing, and identification and localization of post-translational or other covalent modifications [ 17-24]. 

The ionization methods that are used most often to study the peptides and proteins through mass spectrometry are ESI and MALDI. All of these techniques are characterized by the formation of stable ions (because they have only a low excess energy) and the absence of fragments. 

The detection limit of ESI and MALDI depends on several factors, such as the nature of the sample and its preparation and purity, the instrument used and the skill of the operator. For peptides and proteins, the detection limit is in practice somewhere between femtomoles and picomoles, even if attomole limits have been reported [25-27], 

Because the resolution needed to separate different peaks in the isotopic cluster of a small peptide is lower than the resolution of most analysers, the molecular mass that is measured corresponds to that calculated using the predominant isotope of each element. This is not so in the case of proteins. Because the resolution required to resolve the isotopic cluster increases with the mass or with the charge carried by the ion and because the resolution of analysers is limited, the various peaks in the isotopic cluster combine and form a single peak that spreads over several masses (15 Da at 10 000 Da and 45 Da at 100 000 Da) 

Conditions column, TSKgel, G2000SW eluent, 0.15 M phosphate buffer containing 1 M NaCl, 20 % methyl cellosolve and 1 % SDS temperature, 22 °C flow rate, 0.9 mL/min. Reproduced from ref. 54 with permission. 

By this method, the samples could be separated from salt within a short time with only a monor loss of the materials. 

Morimoto et al. [ref. 56] demonstrated that the rapid detection of activated rat urinary kallikrein (Mw 44,000), which was obtained by the trypsin treatment of inactive kallikrein purified from rat urine, could be made by post column reaction. After the SEC separation, the enzyme was allowed to react with polyphenyl-alanylarginine-4-methylcoumaryl-7-amide. In this work, TSKgel G3000SW was used as a column. The results obtained showed that the rapid detection of activated enzyme could be carried out by this technique. 

Cretin et al. [ref. 57] described the purification method of physiologically active phosphoenolpyruvate carboxydase from sorghum leaves. The technique they used was a SEC separation of immuno-precipitates of the enzyme. A highly specific immune serum was used for the immunoprecipitations. They determined that the iso- 

The inhibitory capacity of a serine protease inhibitor, o( -pI [ref. 58], was investigated by Feste and Gan [ref. 59] by use of a SEC separation of the inhibitor-proteinase complex. The protein-ases used in the investigation were trypsin and elastase. A column and an eluent utilized were TSKgel G2000SW and 0.1 M sodium phosphate buffer. In the SEC separation, the molar ratio of the inhibitor to elastase was varied, and the investigators measured the resulting increase in the peak of the complex and a concomitant reduction of the corresponding elastase peak. This increase in the peak was used to assay the inhibitory capacity of o i-pI. The obtained data were compared with those from a conventional spec-trophotometric assay and found to be consistent with them. 

Koshland and J. T. Edsall have recorded their own experiences in early protein research. Koshland stresses the value of his induced fit theory,192 while Edsall describes his long career at Harvard after two years with Hopkins at Cambridge.193 Fruton has also described the work of T. B. Osborne (1859-1928), noted for his analyses of amino-acids from seed proteins.194 The idea that enzymes might be proteins was a matter of heated debate among chemists from about 1915, including arguments between Willstatter and James Sumner, who in 1926 isolated the enzyme urease and showed it to be a protein.195 However, while it is most important to emphasize protein chemistry, the contributions made to protein science by physics 

The role of certain enzymes in cell control and human disease has also received attention, especially in regard to the specificity of enzymes and to protein phosphatases.200 There has also been intense interest in biocatalysis as an application of enzyme chemistry in organic synthesis,201 in the uses of proteolytic enzymes as shown by the work of Perutz,202 and in nucleotide enzymology.203 New methods of investigating the complexities of enzyme action have been described,204,205 as have new methods of determining enzyme structures using studies of bacterial nutrition.206 Other studies have investigated the relations between allosteric proteins and enzymes,207 and the mechanisms by which they function. 

European Company to produce insulin and important suppliers of oestrone,274 Lederle,275 and Roussel Uclaf and Schering AG in Europe.276 Another development arising from this early research on steroids was Barton s pioneering studies in conformational analysis. The reminiscences include those by Fried on the discovery of the fluorosteroids at the Squibb Institute for Medical Research in New Jersey,277 and of studies of the biochemistry of steroid hormones linked to genetics and the history of cancer.278 Clearly, the problems of unravelling the complex mechanisms of steroid action have aroused considerable interest.279 

R Multhauf, The Origins of Chemistry, Gordon Breach, Williston, VT, 1993. 

Balaban, J. Erlen and R. Siderits, eds, The Skilful Physician, Harwood Academic, New York, 1998. 

Several trends can be discerned from this table. 

The majority of these drugs are administered parenterally, either subcutaneously, intramuscularly, or systemically by intravenous injection or infusion. Many of the drugs in this table have very high systemic absorption from subcutaneous and intramuscular dosage forms. 

An example of an inhaled protein is DNase (Pulmozyme). It is an enzyme used to break down thick mucus secretions in the respiratory tracts of patients with cystic fibrosis. An inhaled protein that requires systemic 

Compound Molecular Volume of Elimination half life (h) How administered (systemic Use 0  

Agalsidase beta rh r-galactosidase A (Fabrazyme) 45.4 0.75-1.7 IV injection (1.0) Fabry disease n a 

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The CA Registry of CAS also comprises 1.6 million protein and peptide sequences and over 21.6 milhon nudeic acid sequences from literature sources and from GenBank. 

A purple or rose pink coloration i produced when sodium hydroxide and dilute copper sulphate solution are added to compounds containing two -CONH- groups attached either to one another, or to the same nitrogen atom, or to the same carbon atom. It is therefore also given by oxamide, NHjCO CONH, malonamide, NHtCO-CH, CONH, and by proteins and peptides. In fact the -COKH - is often spoken of as the peptide linkage. 

However, interpretation of, or even obtaining, the mass spectrum of a peptide can be difficult, and many techniques have been introduced to overcome such difficulties. These techniques include modifying the side chains in the peptide and protecting the N- and C-terminals by special groups. Despite many advances made by these approaches, it is not always easy to read the sequence from the mass spectrum because some amide bond cleavages are less easy than others and give little information. To overcome this problem, tandem mass spectrometry has been applied to this dry approach to peptide sequencing with considerable success. Further, electrospray ionization has been used to determine the molecular masses of proteins and peptides with unprecedented accuracy. 

Reversed-phase high performance Hquid chromatography has come into use for estimating the purity of proteins and peptides as weU. However, before employed, a high performance Hquid chromatographic (hplc) profile of a given protein must be completely vaHdated (43). 

E. Rothsteia, ia R. Harrison, ed.. Protein and Peptide Purification Process Eepelopment and Scale-Ep, Marcel Dekker, Inc., New York, ia press. 

The lungs large, permeable surface makes systemic deUvery possible. For example, an inhaler deUvers 360 )Tg per dose of aerosolized ergotamine tartrate [379-79-3] for migraine (54), and inhalant systems deUver anesthetic gases. Research is under way on the systemic deUvery of proteins and peptides through the lungs (57,58). 

Absorption of proteins and peptides, which has been reviewed (60), is generally low and somewhat erratic. The judicious use of absorption enhancers may be necessary and can be accomphshed in a very controUed manner in this area. The mouth is routinely exposed to a wide variety of agents of different pH and osmolarity and appears to be more robust than many other epitheha. Exposure to a wide range of pH values produced damage only at the extremes of... 

The separation of proteins and peptides mixtures is the objective of protein biochemisdy. Albumin (Mr 66 000) concentration in a biological fluid (seaim, urine or cerebrbrospinal fluid) is assayed as markers for a series disease, such as nephritic syndrome or chronic glomuleronephritis. In diabetic patients the progression of microalbuminuria is accompanied by an increase in urinary concentrations of human semm albumen. In normal the excretion of albumin is 20 (tg/ml, in pathology - 20-200 p.g/ml. 

A Caflisch, M Karplus. Molecular dynamics studies of protein and peptide folding and unfolding. In K Merz Jr, S Le Grand, eds. The Protein Eoldmg Problem and Tertiary Structure Prediction. Boston Birkhauser, 1994, pp 193-230. 

Figure 15 Graph of HETP against 1/Dm for 12 Proteins and Peptides...

Porous silica packings do, however, sometimes suffer from adsorption between the sample and silanol groups on the silica surface. This interaction can interfere with the size exclusion experiment and yield erroneous information. In many cases, this problem is easily overcome by selecting mobile phases that eliminate these interactions. In addition, the surface of porous silica packings is routinely modified in order to reduce these undesirable interactions. Trimeth-ylsilane modified packing is typically used with synthetic polymers. Diol modified packing is typically used with proteins and peptides. 

FIGURE 4.7 Separation of mixture of proteins and peptides on a TSK-GEL G2000SWxl column using Wyatt/Optilab DSP and miniDawn detectors. 

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. 

APPLICATION OF SIZE EXCLUSION-HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY FOR BIOPHARMACEUTICAL PROTEIN AND PEPTIDE THERAPEUTICS... 

The specification development process is a data-driven activity that requires a validated analytical method. The levels of data needed include assay precision, replicate process results (process precision), and real-time stability profiles. A statistical analysis of these data is critical in setting a realistic specification. Most often, aggregation and fragmentation degradation mechanisms are common to protein and peptide therapeutics. Therefore, the SE-HPLC method provides a critical quality parameter that would need to be controlled by a specification limit. 

Qnadroni, M., et al., 1996. Analy.sis of global re.spon.ses by protein and peptide fingerprinting of protein.s i.solated by two-dimensional electrophore-.sis. Application to snlfate-starvation re.sponse of Escherichia coli. European Journal of Biochemistry 239 773-781. This paper de.scribes the n.se of tandem MS in the analysis of protein.s in cell extracts. 

Harper, E., and Rose, G. D., 1993. Helix stop signals in proteins and peptides The capping box. Biochemistry 32 7605-7609. 

P. D. Grossman, J. C. Colburn, H. H. Lauer, R. G. Nielsen, R. M. Riggin, G. S. Sittampalam and E. C. Rickard, Application of free-solution capillary electrophoresis to the analytical scale separation of proteins and peptides . Anal. Chem. 61 1186-1194 (1989). 

A. W. Moore-Jr, J. P. Lamiann-Jr, A. V. Lemmo and J. W. Jorgenson, Two-dimensional liquid chromatography-capillary electrophoresis teclmiques for analysis of proteins and peptides . Methods Enzymol. 270 401-419 (1996). 

Proteins and peptides are amino acid polymers in which the individual amino acids, called residues, are linked together by amide bonds, or peptide bonds. An amino group from one residue forms an amide bond with the carboxyl of a second residue, the amino group of the second forms an amide bond with the carboxyl of a third, and so on. For example, alanylserine is the dipeptide that... 

In the context of gene expression analysis, gene products include proximal products, such as primary transcripts, intermediate products, such as mRNA, tRNA, and rRNA, and distal products including proteins and peptides. 

TABLE 2. Proteins and peptides whose biological activity is affected by oxidation of methionine residues ... 

The nasal tissue is highly vascularized and provides efficient systemic absorption. Compared with oral or subcutaneous administration, nasal administration enhances bioavailability and improves safety and efficacy. Chitosan enhances the absorption of proteins and peptide drugs across nasal and intestinal epithelia. Gogev et al. demonstrated that the soluble formulation of glycol chitosan has potential usefulness as an intranasal adjuvant for recombinant viral vector vaccines in cattle [276]. 

Table 5.5 Nomenclature of the ions formed in the mass spectral fragmentation of polypeptides. From Chapman, J. R. (Ed.), Protein and Peptide Analysis by Mass Spectrometry, Methods in Molecular Biology, Vol. 61, 1996. Reproduced by permission of Humana Press, Inc. 

J. R. (Ed.), Protein and Peptide Analysis by Mass Spectrometry, Methods in Molecular Biology, Vol. 61, 1996. 

MALDI-ToF is a technique that allows the molecular weights of proteins and peptides to be determined. It is less susceptible to suppression effects than electrospray ionization and thus is able to be used for the direct analysis of mixtures. In the case of a crude tryptic digest, MALDI-ToF will provide a molecular weight profile of the polypeptides present without the analysis time being extended by the need to use some form of chromatographic separation. 

Why is APCl wholly inappropriate for the study of high-moleoular-weight materials suoh as proteins and peptides ... 

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