Enzyme externally buffered

Rowlett and Silverman used a Brpnsted plot to examine the interaction of external buffers with human carbonic anhydrase II. The buffers act as proton acceptors in the removal of the proton generated by the enzyme-catalyzed reaction. The Brpnsted plot displays a plateau at a value of about 10 for the catalytic rate... 

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).

Figure 1441. Sampling from a fermenter for on-line analysis (after [366]). 1. Direct removal of fermentation broth (analyte A) 2. indirect sampling by ultrafiltration, dialysis, electrodialysis, per-vaporation, providing an analyte A of proportional concentration, normally diluted 3. indirect sampling by extraction of fermentation broth by external buffer 4. in situ measurement by means of an enzyme electrode or using a sterile housing with inserted electrode. F = fermenter, W = waste.

To avoid diffusional limitations it is advisable to assay the enzyme activity under more drastic conditions. Amongst other things, this means increasing stirrer speed to exclude external diffusion, crushing the particles to reduce porous diffusion, increasing the substrate concentration to about > 100-fold of K vi-value to avoid lack of substrate at the center of the particles or adding buffer to avoid pH-shifts. If the reaction rate is increased by any of these means it is likely that diffusional control is operative and can to some extent be reduced or even eliminated. 

By using buffers with sufficient capacity (> 0.05 M) that minimize the dynamic pH-gradients. Occasionally the substrates or products themselves provide such properties so that only the optimal external pH-value has to be adapted. It should not be lower than the pK-value of the weak acid and not lower than the optimum pH of the enzyme [95,96]. 

Alexeev et have examined the inhibition by trifluoro alanine, according to the well-known ability of fluoro amino acids to inactivate PLP-dependent enzymes. They indeed observe an irreversible inactivation and established the structure of the adduct. When the crystals of the protein were soaked in a buffer containing trifluoro alanine, they observed the adduct 10 (Figure 10), whereas when the incubation was carried out in solution, the amide 11, resulting from HF elimination, was obtained. The postulated mechanism implies a decarboxylation of the external aldimine in the first step. This is rather surprising since in the normal mechanism, decarboxylation occurs in the intermediate [I]. 

Immobilization of the enzymes to sohd surface induces stractirral changes which may affect the entire molecule. The study of conformational behavior of enzymes on solid surface is necessary for better understanding of the irtrmobilization mechanism. However, the immobilization of enzymes on alginate beads is generally rapid, and depends on hydrophobic and electrostatic interactions as well as on external conditions such as pH, temperature, ionic strength, and nature of buffer [11,12], Enzymes dena-turation may occur under the irtfluence of hydrophobic interactions, physico-chemical properties of the alginate beads or due to the intrinsic properties of the enzyme. 

---