Proteins, introduction enzymes

The use of mass spectrometry for the analysis of peptides, proteins, and enzymes has been summarized. This chapter should be read in conjunction with others, including Chapter 45, An Introduction to Biotechnology, and Chapters 1 through 5, which describe specific ionization techniques in detail. 

The preceding sections of Chapter 7 have discussed iron-containing proteins and enzymes having a porphyrin ring system. Section 7.9 presents a short introduction to the many non-heme iron-containing proteins and enzymes. Two of these are iron-sulfur proteins (Section 7.9.2) and iron-oxo proteins (Section 7.9.3). 

The simplest case to consider would be one where a plant had been genetically modified to make a new protein, a protein without enzyme activity, such as the introduction of the Bt toxin. In this case, the concept of equivalence is typically applied. The case is made that the chemistry of the unmodified plant and modified plant are likely to be so similar that they can be considered to be equivalent. 

The most versatile method for the introduction of bioactive molecules with terminal NH2 functions such as proteins and enzymes is the coupling via formation of a Schiff base (Fig. 47). It is necessary to oxidise the dextran backbone in the first step to obtain reactive aldehyde moieties. The Schiff base formed between the amine group of the bioactive molecule and the oxidised dextran is usually reduced in a separate step to obtain a stable conjugate. 

Peptides and proteins are not strictly new therapeutic agents, indeed hormones, serum proteins and enzymes have been used as drags ever since the commercial introduction of insulin in 1923. However, significant... 

Synthetic derivatives and analogs of prenyl diphosphates have historically played a key role in defining key featnres of the mechanism of enzymes that ntilize these key intermediates in the isoprenoid pathway. This has also been the case with the investigation of the protein prenyl-transferases. A brief introduction to the protein prenyltransferase enzymes is given along with outlines on the previous use of prenyl diphosphate tools and key aspects of their synthesis. The development of prenyl diphosphate-based FTase inhibitors is described. The use of prenyl diphosphate derivatives as mechanistic and structural probes is next discussed. In particular, the use of fluorinated, isotopically labeled, and photoaffinity derivatives is presented. An overview of the extensive work on the determination of FTase isoprenoid substrate specificity is then given, and the chapter concludes with a section on the development of prenyl diphosphate tools for proteomic studies. 

Several thiols occur naturally for example, skunk secretion contains 3-methyll-butanethiol and cut onions evolve 1-propanethiol, and the thiol group of the natural amino acid cysteine plays a vital role in the biochemistry of proteins and enzymes (see Introduction, p. 2). Primary and secondary thiols may be prepared from alkyl halides (RX) by reaction with excess sodium thiolate (SN2 nucleophilic substitution by HST) or via the Grignard reagent and reaction with sulfur. Tertiary thiols can be obtained in good yields by addition of hydrogen sulfide to a suitable alkene. Thiols can also be prepared by reduction of sulfonyl chlorides (Scheme l).la,2a... 

Walters Options The Alternative Cancer Therapy Book (1993) contains a short introduction to the subject under the section Metabolic Therapies, with the statement that proteolytic (protein-digesting) enzymes are believed to dissolve the walls of cancer cells, and that enzyme treatments are widely used in Europe. More specifically, enzymes are used variously in what are called Issels therapy, in Wheatgrass therapy, in the Gerson diet, in Kelley s therapy, and in Niepa- s therapy (Walters, 1993, pp. 86,147,198,207,216). It is noted furthermore that chranotherapy destroys the body s enzymes, that cooking destroys the enzymes in food, and that enzymes may be involved in the so-called spontaneous remission of cancer (Walters, 1993, pp. 155,201). Also noted, in the work of Hans Niepa-, is that the enzyme bromelain, derived from pineapple roots, will deshield cancer cells, as will beta-carotene (Walters, 1993, p. 222). 

Chemical techniques have also been used to prepare nanotube-encapsulated materials. In this context, carbon nanotubes were oxidatively opened by boiling aqueous nitric acid, then filled with oxides of Ni [145], Co [145], Fe [145], U [145],Mo [146],Sn [147,148],Nd [149],Sm [149],Eu [149],La [149],Ce [149], Pr [149], Y [149], Zr [149], Cd [149], pure metals such as Pd [150], Ag [151], Au [151], proteins or enzymes (e.g. Zn2Cd5-metallothionein, cytochrome c(3), /1-lactamaseI,andDNA complexes) [152,153],andAu2S3 [149],AuCl [151],CdS [149] by wetting techniques (see Fig. 23 also [148,149]).In addition, other capillarity wetting methods, such as heating the elements with open-ended nanotubes, have led to the introduction of the elements Pb, Bi, Cs, S and Se into nanotubes [29-31]. From these studies, it was concluded that only low surface tension substances can be introduced into nanotubes. 

A key category of polypeptides is enzymes, which are biological catalysts that accelerate the rate of biological reactions. A few key enzymes will be discussed. This introduction to proteins and enzymes will focus primarily on simple examples and the primary functions of these complex molecules. The other part of this section will concentrate on chemical methods that allow one to identify the structure of proteins. 

The introduction of diethylaminoethyl and propylsulphonic acid groups onto a non-porous hydrophilic polymer of 2.5 /im in particle diameter produces a pair of materials that can rapidly separate proteins with high resolution and high recovery. The TSKgel DEAE—NPR alW TSKgel SP- NPR resins have an ion-exchange capacity of 0.15 meq/ml of column bed volume and provide an addition to the hunily of TSK resins, the TSKgel DEAE—5PW, and SP-5PW Table 5), now well known for their application in protein and enzyme separations. 

Leger C (2012) Direct electrochemistry of proteins and enzymes an introduction... 

As described earlier, translation of the EPSPS mRNA of plants results in the formation of a protein which has an AJ-terminal extension. The AJ-terminal extension, referred to as the chloroplast transit peptide, is necessary and sufficient for the import of the preprotein by the chloroplast. Once imported by the chloroplast, the transit peptide is cleaved releasing the mature enzyme. As expected, introduction of the EPSPS transit peptide to other protein sequences results in the importation of the fusion protein by the chloroplast. 

Most often proteins are the bacterial biopolymers studied using MALDI MS either from fractions or whole cells. They are not the only isolated cellular biopolymers studied by MALDI, nor the first. Very soon after the introduction of MALDI there were a few reports of the analysis of bacterial RNA or DNA from bacterial fractions. One of the first applications of MALDI to bacteria fractions involved analysis of RNA isolated from E. coli,4 Other studies included analysis of PCR-amplified DNA,5 6 DNA related to repair mechanisms7 and posttranscriptional modification of bacterial RNA.8 While most MALDI studies involve the use of UV lasers, IR MALDI has been reported for the analysis of double stranded DNA from restriction enzyme digested DNA plasmids, also isolated from E. coli.9... 

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