Protein during enzymatically

Formaldehyde fixes proteins in tissue by reacting with basic amino acids— such as lysine,5 7—to form methylol adducts. These adducts can form crosslinks through Schiff base formation. Both intra- and intermolecular cross-links are formed,8 which may destroy enzymatic activity and often immunoreactiv-ity. These formaldehyde-induced modifications reduce protein extraction efficiency and may also lead to the misidentification of proteins during proteomic analysis. 

Recently a simplified process was developed for incorporating l-methionine directly into soy proteins during the papain-catalyzed hydrolysis (21). The covalent attachment of the amino acid requires a very high concentration of protein and occurs through the formation of an acyl-enzyme intermediate and its subsequent aminolysis by the methionine ester added in the medium. From a practical point of view, the main advantage of enzymatic incorporation of amino acids into food proteins, in comparison with chemical methods, probably lies in the fact that racemic amino acid esters such as D,L-methionine ethyl ester can be used since just the L-form of the racemate is used by the stereospecific proteases. On the other hand, papain-catalyzed polymerization of L-methio-nine, which may occur at low protein concentration (39), will result in a loss of methionine because of the formation of insoluble polyamino acid chains greater than 7 units long. 

Numerous undesirable reactions that result in organoleptic, nutritional and functional deterioration may occur in food proteins during processing and storage. These include the non-enzymatic or Maillard reactions, transamidation condensation reactions with dehydroalanine forming crosslinks, and carbonyl amine interactions, all of which may involve the free e-amino group of lysine (11,23). To minimize these reactions a significant volume of work has been done on the protective modification of the e-NH2 of lysine by formylation, acetylation, propionylation (26) or reductive dimethylation (10,11). 

Enzymatic hydrolysis of food proteins generally results in profound changes in the functional properties of the proteins treated. Protein hydrolysates may therefore be expected to fulfil certain of the food industry s demands for proteins with particular, well-defined functional properties. A wide-spread use of protein hydrolysates in food requires, however, a careful control of the taste and functionality of the protein during its hydrolysis and subsequent processing to obtain a reproducible product quality. 

The development of the HPLC method to assay enzyme activities has made it considerably easier to assay a single activity in the presence of others. Thus, attempts to obtain a pure protein during the purification procedure may not be necessary. Since the advent of HPLC to assay enzyme activities, it is possible to stop the purification at a much earlier stage and still assay for a single enzymatic activity. In fact, for some studies, it is even advantageous to assay the activity of interest in the presence of other activities. 

The formation of disulfide bonds in proteins synthesized in vitro can be followed by measuring enzymatic activity or by an increased mobility compared to the reduced protein during SDS-PAGF. This increased mobility arises from the fact that, as disulfide-bonded proteins are intra-molecularly cross-linked, they form a more compact structure and occupy a smaller hydrodynamic volume compared to the reduced protein (Gold-enberg and Creighton, 1984). An illustration of this increase in mobility is shown in Fig. 2. Here the mRNA for preprolactin was translated in a cell-free system optimized for the formation of disulfide bonds, and then analyzed by SDS-PAGF. The translocated protein forms disulHde bonds under these conditions whereas the protein synthesized under the same conditions but in the absence of microsomal membranes does not form disulfide bonds. Thus the nascent protein must be translocated into microsomal vesicles for disulfide bond formation to occur. 

Organophosphates are active not only against acetylcholinesterase but also serine proteases - which is obviously due to the shared catalytic mechanism. DFP is actually being used as a protease inhibitor in biotechnology. Another inhibitor that shares its mode of action but is less dangerous (because it is not volatile, and the enzyme adducts it forms are less stable) is PMSF (phenylmethylsulfone fluoride). You may have encountered it in one or the other research lab it is commonly added to crude cell extracts in order to minimize enzymatic breakdown of proteins during purification. 

Figure 4 A schematic representation of the experimentai approach for time-resoived XAS measurements. XAS provides local structural and electronic information about the nearest coordination environment surrounding the catalytic metal ion within the active site of a metalloprotein in solution. Spectral analysis of the various spectral regions yields complementary electronic and structural information, which allows the determination of the oxidation state of the X-ray absorbing metal atom and precise determination of distances between the absorbing metal atom and the protein atoms that surround it. Time-dependent XAS provides insight into the lifetimes and local atomic structures of metal-protein complexes during enzymatic reactions on millisecond to minute time scales, (a) The drawing describes a conventional stopped-flow machine that is used to rapidly mix the reaction components (e.g., enzyme and substrate) and derive kinetic traces as shown in (b). (b) The enzymatic reaction is studied by pre-steady-state kinetic analysis to dissect out the time frame of individual kinetic phases, (c) The stopped-flow apparatus is equipped with a freeze-quench device. Sample aliquots are collected after mixing and rapidly froze into X-ray sample holders by the freeze-quench device, (d) Frozen samples are subjected to X-ray data collection and analysis.

Extraction of G proteins from membranes usually enhances ADP-ribosylation. In addition, small amounts of detergents like SDS, CHAPS or Lubrol PX facilitate the toxin s enzymatic activity (see above). For detection and identification of G proteins during G protein purification procedures, aliquots are also subjected to PT-catalyzed ADP-ribosylation for detection and identification (Rosenthal... 

Table I. Application of Membrane Processes During Enzymatic Modification of Proteins...

Depending on external conditions (pH, temperature, composition of solution) and molecular structure (changes of protein structure upon natural or artificial mutations), equilibrium between different conformers can be dynamic. During enzymatic reaction the excited LO (LO ) is generated in the active site of each conformer. Initially there is a mixture of complexes ... 

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