The Enzyme Sample

A final point to be considered in the use of HPLC as an assay procedure is the enzyme itself. Will the activity be a pure enzyme Will it be part of a rather crude cell-free extract Or will it be present in a fermentation broth In the latter two cases, the presence of contaminating activities must be considered. While as mentioned above, these activities, by affecting the recovery of the product or even by affecting substrate levels during the course of the reaction, could easily cause problems with other assay procedures, they are not a problem for the HPLC assay method. Thus, HPLC should be considered first when activity in some crude extracts is to be assayed. 

The assay of the activity of an enzyme can be subdivided into several steps formation of a reaction mixture, preparation of an enzyme sample, combination of the two to initiate the reaction, incubation of the reaction, termination of catalysis, separation of components, their detection, and finally, reduction or processing of the data. 

Not all assays require a separation step, and this fact may be used to develop a classification scheme for assay methods. Assays that require no separation have been grouped under the heading continuous assay methods, while discontinuous methods incorporate those that do. 

The need to use a discontinuous assay method does not automatically mean that the HPLC method is the procedure of choice. For HPLC to be suitable, it must be possible to separate the components, and some method for detection and quantitation must be available. Next, neither the ingredients in the reaction mixture nor those used to terminate the reaction should produce problems for the separation and detection. Finally, the enzyme itself should be considered. 

Excess protein can contaminate columns, and extraneous enzymes can cause problems in quantitation. 

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Fig. 4. Anaerobic titration of xanthine oxidase with xanthine at pH 8.2 with a reaction time of 2 min. at about 20°. The integrated intensity of the Rapid molybdenum EPR signals (in arbitrary units) is plotted against the number of moles of xanthine added per mole of active enzyme. Activity/A4jo for the enzyme samples used was 112 corresponding to an active enzyme content of 57%. Thus the molar ratios of xanthine/total xanthine oxidase have been multiplied by 1.76 to refer to the active form only. Some of the EPR spectra (recorded at about — 130° and 9.3 GHz) are reproduced to show the changes in signal type as the amount of xanthine is increased. (Data re-calculated from ref. 88, with intensities corrected for variations in tube diameter and enzyme concentration calculated in terms of active enzyme.)...

The relative remaining activity of alcohol dehydrogenase in an optimal water-ethanol mixture exceeded 100%. The observation of the improved activity of some of the enzyme samples after this procedure [179] was a precursor to later achievements on enzyme activation in organic media [178],... 

To study the effect of hydrogen peroxide, the lignin peroxidase was incubated in buffer, either pH 3.0 or 5.0, at the temperature of either 0°C or 10°C. Protein concentration was 30/i,g/ml and the concentration of H2O2 was 0.2 -11.6 mM. The incubation time varied between 0 and 295 min. After incubation 0.4 ml of the enzyme sample was pipetted directly into the activity assay mixture. 

Plot the four relative fluidities (FR) as the ordinate against the four reaction times (7 N) as the abscissa. This should result in a straight line. The slope of the line corresponds to the relative fluidity change per minute and is proportional to the enzyme concentration. The slope of the best line through a series of experimental points is a better criterion of enzyme activity than is a single relative fluidity value. From the curve, determine the PR values at 10 and 5 min. They should have a difference in fluidity of not more than 0.22 and not less than 0.18. Calculate the activity of the enzyme sample as follows ... 

Enzyme Sample Dilute the enzyme samples in MES Buffer with Brij 35 and Sodium Chloride such that the absorbance value of Sample HI of each sample lies within the absorbance values of the Acetoin Standard curve. 

Procedure Preheat the Enzyme Sample solutions, the MES Buffer, and the a-Acetolactate Substrate in a 30° water bath for approximately 10 min. Prepare the following solutions for each enzyme sample in the following order ... 

Enzyme Blank B1 Pipet 200 pL of the Enzyme Sample solution into a 10-mL test tube in the 30° water bath. Add 200 pL of MES Buffer, mix, and immediately replace the test tube into the water bath. 

Assay since one unit of SOD activity is defined as the amount of enzyme that inhibits by 50% the rate of reduction of cytochrome c under specified conditions, it is necessary to try several different dilutions of the enzyme preparation. Solution B should be kept at 4°C and solution A warmed to 25°C a thermostatted spectrophotometer cell at 25°C should be used at 550 nm for maximum sensitivity the spectrophotometer should be set to the observed maximum absorbance when a portion of solution A is reduced with a few crystals of dithion-ite. 2.9 ml of solution A is then placed in a 3 ml cuvette and 50 /A of the enzyme sample is added with mixing. The reaction is started by adding 50 /A of solution B with further mixing and the change in absorbance at 550 nm is monitored. The enzyme sample should be replaced by water or by several standard SOD solutions to obtain a blank value, which should be subtracted, and a range of standard curves. Plots of l/AE min-1 for the standard enzyme are used to determine the activity of the unknown enzyme preparation the JE min 1 value is obtained from the linear part of the curve. 

The reaction mixture contained N-acetylglucosamine, UDP-Gal, MnCh, and buffer at pH 8.0. The reaction was started by the addition of the enzyme. Samples were transferred at intervals to cacodylate buffer on ice (pH 6.5) to terminate the reaction. Samples (10 /u.L) were analyzed by HPLC. The conversion of UDP-Gal to UDP is shown in Figure 9.68. Each panel represents a different time point, from 0 to 60 minutes. During the incubation, the disappearance of the substrate and the formation of the two products is seen. The enzyme was obtained from commercial sources and human serum. 

The reaction mixture contained cyclic formycin monophosphate, an analog of cAMP, as the substrate, Tris-HCl (pH 7.5) as buffer, and MgQ2. The reaction was started by the addition of the enzyme. Samples were removed at intervals and injected directly onto the reversed-phase column for analysis. Figure 9.108 shows chromatograms after 10 and 30 minutes of incubation. While the amount of cFoMP substrate in the incubation mixture has declined and the amount of product FoMP has increased, the amount of formycin A (FoA), the analog of adenosine, has remained unchanged. When the area of each peak is plotted as a function of reaction time, the data shown in the central inset are obtained. Although these data clearly illustrate the activity of the cyclic phosphodiesterase, they also show the absence of any 5 -nucleotidase. 

Figure 3. Analytical disc gel electrophoresis of purified cellobiase in high and low pH gel systems. A total of 35 pgrams of purified cellobiase was used as the enzyme sample in each gel. The gels were stained for protein with Coomassie blue. The right-hand gel was obtained at a running pH of 4.5 toward the cathode and the left-hand gel at a running pH of 9.5 toward the anode (49).

Figure 4, Analytical disc gel electrophoresis of the hydrocellulases A, B, and C. Hydrocellulases A, B, and C were run in gels A, B, and C respectively. A total of 30 fjigrams of protein was used as the enzyme sample in each gel. Gel D is the pattern obtained when 100 ixgrams of water fraction protein was used as the sample. Gels were stained with Amido-Schwarz (53).

The filter paper activity of the enzyme samples was determined at 50 °C according to standardized NREL filter paper assay [11]. Carboxymethylcellulase (CMCase endoglucanase) and Avicelase (exoglucanase) activity was determined by measuring the release of reducing... 

DTNB reacts not only with thiocholine but also with other thiol compounds, thus producing TNB. When cholinesterase samples contain other thiol compounds, as in hemolyzed erythrocytes, DTNB should be added to the enzyme sample before the substrate. When the reaction of DTNB with the other thiols is completed, the substrate is added. 

Thiocholine esters react with oxime groups (oxymol-ysi.s), whereby thiocholine is produced (Primoiic et ai., 2004 Skrinjaric-Spoljar et al., 1992). When samples contain oximes, the contribution of oximolysis should be measured in the absence of the enzyme and subtracted from the total thiocholine increase measured in the enzyme sample. However, when blood samples are taken from patients under oxime therapy, the samples may contain an unknown oxime concentration, and one has to be aware that due to oximolysis, the measured increase in thio-cholinc may lead to a false conclusion concerning enzyme activity. 

The fourth step in the direct ethanol process is considered to be key to the economic viability of the bioconversion of cellulose to chemicals. The pretreated cellulose slurry is simultaneously converted to glucose and the glucose to ethyl alcohol in the same vessel in a continuous or semi-continuous mode. The enzyme sample is the whole culture from the enzyme production vessel. The feedstock is a slurry of 7.5% to 15% cellulose. The yeast is either added as a cake or recycled as a cream. 

In the measurement of enzyme activity, a high substrate concentration that is greatly in excess of the Km value is always used, and the enzyme sample to be investigated is correspondingly diluted vmder the conditions, the rate of the enzyme-catalyzed reaction depends only on the enzyme concentration, i.e., it is a zero order reaction. Even under conditions of substrate saturation, the measured catalytic activities are influenced by slight differences in reaction conditions, such as the temperature, composition and concentration of the buffer, pH value, nature of the substrate and its concentration, coenzymes, and protein content in the sample. Therefore, the results of measurement of the catalytic activity of an enzyme are in principle method dependent direct comparison of the results between laboratories is made difficult by the use of different methods in different laboratories. 

Fig. 4A-D. Digestion of poly(ADP-ribose) synthetase with chymotrypsin or papain after modification of 3-(bromoacetyl)pyridine in the presence and absence of NAD. The enzyme sample (25 jug), chemically modified in the presence ( ) and absence (O) of NAD and labeled with NaB H4, was digested with 0.5 Mg of a-chymotrypsin (left) or 0.5 jug of papain (right) and subjected to SDS gel electrophoresis. A, C Quantification of the radioactivity in SDS-gel B, D the difference between the values denoted by and O in A and C was replotted...

Second, the replacement of the native activating cations (e.g., by natural Co under identical conditions) ideally should not result in an irreversible deactivation of the enzyme. In the latter case, the correspondence between the Co" form in the enzyme sample under study and the cobalt(ll) form in the physiologically active enzyme would be doubtful. 

Finally, the quantity of the substituted Co + should conform to the overall number of the cation-binding sites in the enzyme sample. Any excessive Co +, binding to different functional groups of the protein beyond the active centers, would evidently lead to an unpredictable complication of the spectra. 

Fig. 3. Molecular heterogeneity of human PP-ribose-P amidotransferase demonstrated by gel filtration. Two milliliters of the enzyme sample were applied to an 8% agarose column equilibrated with 50 mM potassium phosphate buffer, pH 7.4 containing 5 mM MgCl2> 60 mM -mercaptoethanol, and 0.25 M sucrose. (From Holmes, et al., in press).

A possibly more serious problem concerns recent EXAFS data on xanthine oxidase reported by Tullius et al (1979). In principle, the EXAFS method is capable of determining the nature of the ligand atoms in xanthine oxidase and of providing estimates of their distances from molybdenum. These workers suggested that there were two terminal oxygens in the enzyme but no terminal sulfur. Unfortunately, however, the enzyme sample which they used was a mixture of the functional and desulfo forms, and no attempt was made to separate the EXAFS spectra of the two species. One wonders whether the spectrum of desulfo xanthine oxidase could have been dominant. 

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