Enzyme activity measurement, effect denaturants

Folding Equilibrium Studies. E. coli RTEM p-lactamase is a monomeric protein. Its amino acid sequence has been determined (79). It has one disulfide bond between the residues Cys S and Cys. The presence of four tyrosines and four tryptophans allows the use of spectroscopic method for the conformational characterization of the enzyme. In this study, the effect of denaturants on the unfolding of p-lactamase was determined from activity measurements, difference spectroscopy and fluorescence intensity measurements. 

It has been observed that the rate of enzyme reaction rises with temperature up to a certain maximum above which, thermal inactivation of the enzyme takes place. The inactivation of enzymes by heat is due to the denaturation of the enzyme protein. The effect of the instability of the enzyme, free and in the immobilised state, can be studied by exposing the enzyme to thermal treatment for a defined period prior to measuring its activity at a temperature at which it is stable. Chaubey and co-workers obtained 40 °C as the critical temperature of PPy-polyvinyl sulfonate films immobilised with crosslinked lactate dehydrogenase [123]. The activation energies below and above the critical temperature were found to be 93.3 and 22.4 kj/mole, respectively. 

The rate of hydrolysis of DNA, RNA, and polynucleotides can be measured by a sensitive spectrophotometric assay which is based on the hyperchromicity that occurs upon hydrolysis of these substrates (S). The enzyme has a 7-fold greater affinity for denatured DNA than for RNA (8). No inhibitory products accumulate during the course of the reaction. The pH optimum for RNase and DNase activities is between 9 and 10, depending on the Ca2+ concentration. At higher pH values less Ca2+ is required. The inhibitory effect of high Ca2+ observed consistently by many investigators is more pronounced at higher pH values (S). 

Fig. 1. The effect of hsp70 on the refolding of cAAT and pmAAT. Refolding of acid unfolded cAAT or pmAAT was performed by rapid dilution of the denatured enz3rmes in the refolding buffer to a final protein concentration of 1.8 pM. When present, hsp70 (1.8 pM) was added to the refolding buffer before initiation of the refolding reaction. After incubation for 120 min at 10 C, the transaminase activity recovered was measured as indicated under Methods. Reactivation data are expressed relative to that of the native enzyme incubated under identical conditions.

Chemical studies also support the indicated mechanism. For example, the p-oxoacid intermediate formed in step h of Eq. 13-48 or Fig. 13-12 has been identified as a product released from the enzyme by acid denaturation during steady-state tumover. Isotopic exchange with in the solvent and measurement of C isotope effects have provided additional verification of the mechanism. TTie catalytic activity of the enzyme is determined by ionizable groups with piC values of 7.1 and 8.3 in the ES com-plex. ... 

Rat pancreas amylase activity was found to be irreversibly destroyed when subjected to polyacrylamide gel electrophoresis.The measure of enzyme inactivation was dependent on the duration of electrophoresis. In the presence of urea the amylase denaturing effect was enhanced. 

Virtually all enzymatic assays are carried out at 20-50 °C in aqueous buffers of known pH and controlled composition. Both temperature and buffer properties affect the rates of enzyme- catalyzed reactions markedly. The effects of temperature can usually be summarized by a bell-shaped curve (Fig. 4 A). At lower temperatures, reaction rates increase with temperature, but beyond a certain point, denaturation (unfolding) of the enzyme molecules begins, so they lose their ability to bind the substrate, and the reaction rate falls. The temperature giving maximum activity varies from one enzyme to another, according to the robustness of the molecule. In some cases, it may be convenient to use a temperature rather below this maximum, otherwise the rate becomes too high to measure precisely. The rates of many enzyme-catalyzed reactions increase by a factor of ca. 2 over a range of I0°C in the region below the maximum of the... 

The most convenient and comparable measurement of enzyme stabUity is the half-life at a given temperature (other incubation conditions being defined), i.e. the time period over which half the initial activity of an enzyme is lost. Additional information on the effect of temperature on activity can be important, for instance the shape of a plot of logio of percent activity remaining versus time of incubation can indicate whether irreversible thermal denaturation is the sole eause of loss of activity or whether additional factors are involved, such as autolysis, renaturation, or substrate effects. 

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