Buffers enzymes

The components listed in Table 20-1 are typical for PCR. As can be seen, it is a fairly simple reaction to set up. The template and primer concentrations must be determined beforehand the buffer, enzyme, and deoxynucleotide triphosphate (dNTP) mixture are commercially available. 

Drawing on this and the reported specificity of tannin-protein interactions ( ) leads to the conclusion that any useful in vitro modelling of the impact of tannins on digestion must consider more than pH and the concentrations of the buffer, enzyme, substrate, and tannin. The actual enzyme-substrate system must be nutritionally realistic to control for specificities of the reaction of tannins with proteins (including enzymes) gastrointestinal mucoproteins should perhaps also be included on the same grounds. Besides all this, misleading results nay still be obtained if bile surfactants are omitted from the equation. 

Nowadays a wide variety of food ingredients are already produced in an encapsulated form. These comprise artificial sweeteners (aspartame), flavouring agents such as oils or spices (with desirable flavour but possibly undesirable odour), natural colorants (e.g., p-carotene, turmeric), preservatives, acids (citric, lactic and ascorbic), bases, buffers, enzymes, lactic acid bacteria, and some antioxidants (Kirby, 1991 Gibbs et al, 1999 Chen et al, 2006b Ubbink and Kruger, 2006 Augustin and He-... 

It is interesting to note that the subcellular distribution of the thermodynamic buffer enzymes is also in accord with their physiological... 

An overview concerning the enzyme-based electrochemical biosensors published during the last 5 years for heavy-metal determinations is reported. Their sensitivity and selectivity toward inhibitors and the factors (pH, buffer, enzyme, and inhibitor concentrations) affecting the analytical characteristics of these biosensors are also discussed. 

Worms washed to remove free label. Homogenised in buffer + enzyme inhibitors + Na deoxycholate. Extracted at 4°C and centrifuged. Radioactivity counted... 

Table 5.6 Specific activity data for amylase activity after heat treatment of buffered enzyme at 90°C...

Usually restriction enzymes are supplied with the adequate 10X reaction buffer. Enzyme concentration and reaction buffer composition may vary from one source to another, and volumes need to be adjusted accordingly. 

It could be shown, moreover, (Vallee and Neurath, 1955) that five times recrystallized carboxypeptidase was completely inhibited by metal chelating agents, such as 8-OHQ-5SA and 1-10 phenthroline at concentrations of 10 W,Q ,Q Dat concentrations of 10" Af, and some 30% byEDTA at 10 M. These are all known to form complexes with zinc in simple systems. In these experiments, the buffered enzyme solutions were incubated with the chelating agent at pH 7.5, 4°C., for 1 hour prior to the addition of the substrate. Inhibition did not occur when these chelating agents were first incubated with an equimolar amount of zinc, cupric, or ferrous ions. Sodium diethyldithiocarbamate, zincon, sulfanilamide, and diamox, the latter two employed because of their effect on carbonic anhydrase, had little, if any, effect on carboxypeptidase activity. DPN, nicotinamide, and A-methylnicotinamide, examined because of their effect on the ADH sys-... 

In an externally buffered enzyme electrode (Fig. 45), substrate-free buffer is continuously pumped between the dialysis membrane and the enzyme layer (Cleland and Enfors, 1984), i.e., the sample is diluted before it reaches the enzyme. The intensity of the buffer flow may be used to adjust the measuring range and sensitivity. The configuration of the sensor permits it to be sterilized. While the membrane is protected by continuously flowing buffer, the rest of the sensor can be sterilized for 1 h in a solution of 95% ethanol and 5% H2SO4. 

Fig. 45. Externally buffered enzyme electrode. (Redrawn from Cleland and Enfors, 1984).

Assay Buffers and Solutions Besides critical reagents and microtiter plates, the third major component of an ELISA method comprises assay buffers and solutions, including coating buffer, blocking solution, assay diluents, wash buffer, enzyme and substrate, and stop solution. 

Time dependence of 615 5 and e,8.i of cobalt(II)-substituted CA III after addition of CO2 to a buffered enzyme solution at pH 8. The initial drop of absorbance reflects the accumulation of a CoOHj intermediate. ... 

Degradation by enzymes is carried out generally by incubation of the samples with buffered enzyme solutions with suitable blanks to correct for buffer degradation and contamination proteins and/or degradation of the enzymes (II). The degrees of degradation of samples can be followed by methods (e-h) mentioned above. 

Add grain extract and buffered enzyme to test tube... 

The PL decay time was measured 5 min after addition of fhe analyte to the buffered enzyme solution. We note that the oxidation reactions of the above analytes were slow and, as expected, depended on the enzyme concentration. As seen, 1/x vs. the ethanol concentration is linear over a concentration range of 0-0.3 mM. 

The lack of simple spectroscopic handles for the CA systems has made kinetic studies somewhat difficult. In one study, the C linebroadening method has been used. The most common technique is the stopped-flow indicator method in which a solution of CO /HCOj is mixed with a solution of buffer, enzyme and indicator. The small pH change caused by formation or loss of due to reaction (8.22) can be monitored spectrophotometrically. More recently, the method of choice has been to use 0-enriched COj/HCOj" in combination with a mass... 

Glass microchip with Y-junctions at the inlet and at the outlet, continuous flow operation n-hexane and substrates and buffered enzyme solution fed from each inflow buffer and n-heptane recovered in the Y-shaped outlet... 

This is most usually achieved with buffers. Enzyme reactions, electrophoretic separations and also spectrometric and electrochemical determinations may aU require pH control. 

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