Other Proteins

Proteins other than enzymes have been targeted with DCC systems on a number of occasions. As is the case for enzymes, mainly easily accessible (commercially available) proteins have been used, whereas in some instances proteins of more precious character have been challenged with the technique. 

Concanavalin A (Con A), immobilized on solid-support, a bis-maimoside stracture was selected. Generation of the library was performed at neutral to slightly basic pH, where the redox exchange is rapid, and the exchange could efficiently be stopped by lowering the pH. 

Acyl hydrazone exchange has also been probed as reversible chemistry for the generation of oligotopic and directional carbohydrate DCLs of larger size [45]. 

Another account describes a similar system, where 1-thiosaccharides and cysteine-containing oligopeptides were allowed to form prototype disulfide glycopeptide DCLs [47]. In this case, wheat germ agglutinin was instead targeted and 

Highly intriguing accounts on enzymatic DCL generation applied to a carbohydrate-binding protein have also been reported [50, 51]. Using the N-acetyl-neuraminic acid aldolase (sialic acid aldolase), reversible aldol addition of three or four different carbohydrates to pyruvate was catalyzed, yielding small DCLs of 

We now turn our attention to nonredox proteins. SERS spectra of IgC adsorbed on roughened Ag electrodes were obtained by Grabbe and Buck [289]. The spectra 

Microperoxidase is the heme-containing peptide portion of Cyt c that retains peroxidase activity. Several microperoxidases are available with different numbers of amino acid residues. The conformation of microperoxidase-11 (the microperoxidase with 11 amino acids in the peptide) adsorbed on roughened Ag electrodes was studied using Fourier transform SERS and shown to be adsorbed via the a-helical polypeptide chain [299]. As expected the characteristic amide 1 and 111 bands for the protein backbone were the strongest. Similarly microperoxidase-8 was studied by combined SERRS and electrochemistry where it was shown that the heme existed in the penta-coordinated state and could bind cyanide as the sixth ligand [300]. 

The importance of the functional layer on an electrode to the interaction of an enzyme with the surface is brought out by Kudelski s studies with Cu-containing tyrosinase (a phenol oxidase) [305] and laccase [306]. Using w-functionalized thiols he showed that electron transfer was not prevented between the electrode and the enzyme. That the local environment of the enzyme is important has also been demonstrated in a recent study of human sulfite oxidase using SERRS and cychc 

A detailed discussion of the diverse interactions of proteins with heparin/HS would fall outside the scope of tliis chapter the reader is referred, in particular. 

The studies of chloride binding to hemoglobin, discussed above, have indicated that the binding sites for anions may be well defined in structural terms. A similar approach will probably prove fruitful for the identification of ligand binding sites in other proteins. 

Five proteins which have been systematically studied were chosen for a rather detailed survey in Subsections 8.4.1 - 8.4.5 in order to illustrate the types of information provided by halide ion quadrupole relaxation investigations of protein solutions. In addition, the following proteins have been studied by this method a-chymotrypsin [430] ( Cl), [34] ( Br), [434] ( Cl), [64] ( Cl, Br), [431] ( Cl), 

Since in general these studies are not detailed and do not provide significant methodological aspects in addition to those already presented above, an extensive discussion of the results and their implications is not motivated. However, we will briefly mention the problems considered in these studies as well as the results obtained. 

Zeppezauer et al [34] introduced Br NMR for studies of protein solutions and demonstrated for a number of proteins that halide ion binding independent of metallic sites may be conveniently studied by the halide quadrupole relaxation method. In Ref. [34] also the usefulness of the isotope effect to distinguish between rapid and slow exchange conditions was suggested. 

Recent studies by Nome et al [431 441] have further demonstrated the general applicability of the halide quadrupole relaxation method and have in particular attempted to develop methods to distinguish between different types of anion-protein interactions. The general ideas were mentioned in Section 8.4.2 in connection with the discussion of the nature of the anion binding site(s) of alcohol dehydrogenase. 

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Asher S A and Chi Z H 1998 UV resonance Raman studies of protein folding in myoglobin and other proteins Biophys. 

The avidin-biotin complex, known for its extremely high affinity (Green, 1975), has been studied experimentally more extensively than most other protein-ligand systems. The adhesion forces between avidin and biotin have been measured directly by AFM experiments (Florin et al., 1994 Moy et al., 1994b Moy et al., 1994a). SMD simulations were performed on the entire tetramer of avidin with four biotins bound to investigate the microscopic detail of nnbinding of biotin from avidin (Izrailev et al., 1997). 

The last part of this account will be devoted to protein kinases and protein phosphatases and some recent results we have obtained for them. Protein kinases and phosphatases are signaling biomolecules that control the level of phosphorylation and dephosphorylation of tyrosine, serine or threonine residues in other proteins, and by this means regulate a variety of fundamental cellular processes including cell growth and proliferation, cell cycle and cytoskeletal integrity. 

Glucocorticoids have been shown to inhibit gene transcription of other proteins involved in the inflammatory process, including the key inflammation mediators called cytokines (IL-1, IL3—6, IL8, GM-CSF, TNFa) (10,58,63—65). Steroids have been also shown to suppress the formation of cytokine receptors (10) dexamethasone, in particular, downregulates gene transcription of angiotensin II type 2 receptors (66). 

Minerals, particularly Bentonite, ate used to remove proteins that tend to cause haze in white wines. The natural tannin of ted wines usually removes unstable proteins from them. Excess tannin and related phenols can be removed and haze from them prevented by addition of proteins or adsorbents such as polyvinylpyttohdone. Addition of protein such as gelatin along with tannic acid can even be used to remove other proteins from white wines. Egg whites or albumen ate often used to fine ted wines. Casein can be used for either process, because it becomes insoluble in acidic solutions like wines. 

Table 11. Nutritive Value of Leaf Protein Concentrates and Other Protein Products ...

Products prepared in this manner typically contain significant quantities of coagulation Factors II, VII, and X and other proteins as well as Factor... 

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. 

Many proteins frequendy require the assistance of other protein molecules called molecular chaperonins, for assuming the fine tertiary stmcture in vivo. In E. coli, two such chaperonin molecules bind transientiy to newly synthesized polypeptide monomers, preventing them from aggregating prematurely, until the polypeptides attain their folded state (10). 

Since the acetal exists in equiUbtium with the aldehyde, it is possible for the aldehyde to be released when water is added in a mixed drink, changing the balance and giving a burst of freshness to a mixed drink. Ethyl esters of terpene alcohols in citms oils and other botanicals, plus the ethyl esters of fatty and volatile acids, are formed during prolonged exposure to ethyl alcohol. Certain beverage alcohol products that need to contain milk, eggs, or other protein containing materials must be developed carefully and the added flavors must be considered to prevent the precipitation of the protein and separation of the product. 

Purification. Hemoglobin is provided by the red blood ceU in highly purified form. However, the red ceU contains many enzymes and other proteins, and red ceU membranes contain many components that could potentially cause toxicity problems. Furthermore, plasma proteins and other components could cause toxic reactions in recipients of hemoglobin preparations. The chemical modification reactions discussed herein are not specific for hemoglobin and may modify other proteins as well. Indeed, multifimctional reagents could actually couple hemoglobin to nonhemoglobin proteins. 

Nutritional Requirements. The nutrient requirements of mammalian cells are many, varied, and complex. In addition to typical metaboHc requirements such as sugars, amino acids (qv), vitamins (qv), and minerals, cells also need growth factors and other proteins. Some of the proteins are not consumed, but play a catalytic role in the cell growth process. Historically, fetal calf semm of 1—20 vol % of the medium has been used as a rich source of all these complex protein requirements. However, the composition of semm varies from lot to lot, introducing significant variabiUty in manufacture of products from the mammalian cells. 

Scale-up in fixed-bed reactors is limited by the maximum size of the matrix that can be manufactured as a monolith. Hence, this system is appHcable for small- to medium-scale production of antibodies and other proteins, usually for the diagnostic market. This system has been described in greater detail ia the Hterature (22). 

Other Proteins. Groundnut, fish, and cottonseed proteins have been used to a limited degree in dairy substitutes. The properties of the materials are discussed in thehterature (5). 

Acid Dyes. These are anionic dyes, usually containing sulfonic acid groups, that are substantive to wool, other protein fibers, and polyamides when dyed from an acidic dyebath. The lower the pH the more rapid the dyeing, and exhaustion efficiency is enhanced by increased acidity. 

Enzymes, like other proteins (qv), are composed of up to 20 different amino acids (qv). They accelerate hundreds of reactions taking place simultaneously in the cell and its immediate surroundings, and are essential for the development and maintenance of life. 

Effect of Temperature and pH. The temperature dependence of enzymes often follows the rule that a 10°C increase in temperature doubles the activity. However, this is only tme as long as the enzyme is not deactivated by the thermal denaturation characteristic for enzymes and other proteins. The three-dimensional stmcture of an enzyme molecule, which is vital for the activity of the molecule, is governed by many forces and interactions such as hydrogen bonding, hydrophobic interactions, and van der Waals forces. At low temperatures the molecule is constrained by these forces as the temperature increases, the thermal motion of the various regions of the enzyme increases until finally the molecule is no longer able to maintain its stmcture or its activity. Most enzymes have temperature optima between 40 and 60°C. However, thermostable enzymes exist with optima near 100°C. 

Like other proteins, enzymes are potential allergens. In addition, proteases may act as skin and eye irritants. However, during the production and handling of industrial enzymes, the occupational health risks entailed by these properties can be avoided by protective measures, and by the form in which... 

The numerous separations reported in the literature include surfactants, inorganic ions, enzymes, other proteins, other organics, biological cells, and various other particles and substances. The scale of the systems ranges from the simple Grits test for the presence of surfactants in water, which has been shown to operate by virtue of transient foam fractionation [Lemlich, J. Colloid Interface Sci., 37, 497 (1971)], to the natural adsubble processes that occur on a grand scale in the ocean [Wallace and Duce, Deep Sea Res., 25, 827 (1978)]. For further information see the reviews cited earlier. 

Enzymes are excellent catalysts for two reasons great specificity and high turnover rates. With but few exceptions, all reac tions in biological systems are catalyzed by enzymes, and each enzyme usually catalyzes only one reaction. For most of the important enzymes and other proteins, the amino-acid sequences and three-dimensional structures have been determined. When the molecular struc ture of an enzyme is known, a precise molecular weight could be used to state concentration in molar units. However, the amount is usually expressed in terms of catalytic activity because some of the enzyme may be denatured or otherwise inactive. An international unit (lU) of an enzyme is defined as the amount capable of producing one micromole of its reaction product in one minute under its optimal (or some defined) reaction conditions. Specific activity, the activity per unit mass, is an index of enzyme purity. 

The affinity method may be biospecific, for example as an antibody-antigen interaction, or chemical as in the chelation of boronate by ci5-diols, or of unknown origin as in the binding of certain dyes to albumin and other proteins. 

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