Estimating Enzymatic Activities

Though it was realized very early that microdialysis can serve as an excellent tool for estimating in vivo enzymatic activities (Sharp et al., 1986), this application remained relatively unexplored until recently (Westerink et al., 1990). Microdialysis offers several advantages over existing in vitro techniques because it affords the opportunity for continuous sampling from organs other than blood and because of its high spatial resolution. 

---

Based on the rate at which novel microorganisms are being discovered, one can make an estimation of the amount of different organisms that have been identified up to now relative those that remain uncharacterized (Table 5.8). The overwhelming biochemical diversity present in nature makes that the isolation of microorganisms with novel enzymatic activities is worthwhile (reviewed in Bull, Goodfellow Howard Slater, 1992). Preferred sites to find novel enzymes include so-called mega-diversity countries such as Mexico, Colombia, Brazil or India and biodiversity hot spots like rain forest and deep seas. 

Competitive Inhibition Enzyme Assays. Estimates of antibody-lysozyme dissociation constants can be obtained by taking advantage of the fact that most monoclonal antibodies efficiently inhibit enzymatic activity.3 5 The combining site of HyHEL-10, which is presented as an example, also has been demonstrated by X-ray crystallography to overlap a portion of the catalytic site of lysozyme.7 A constant concentration of lysozyme is incubated with varying amounts of antibody, and amounts of free (unbound) lysozyme molecules are estimated by the proportion of catalytic activity remaining. The assay assumes that the addition of Micrococcus lysodeikticus cell walls and concurrent dilution of the antibody-antigen mixture do not disturb the equilibrium. 

A less complex approach can be used for enzyme activity estimation namely, fractions can be collected and enzymatic activity measured in these fractions. The practical realization involves a normal run before fraction collection to detect the migration time of the peak, which should be collected. The time Tc when the peak is going to leave the capillary can be estimated as retention time multiplied by the length of the capillary divided by the capillary length to the detector. When fractions are to be collected, the potential is switched off just before Tc. A new vial is placed at the end of the separation... 

Various methods have been used to estimate water-column denitrification rates including stoichiometric relationships, measurements of the enzymatic activity of the electron transport system (ETS), and N03 incubation experiments. In combination with residence times or mass transport calculations these measurements yield areal denitrification rates. 

Protonation State. At optimal pH for enzymatic activity ( 5-6) [101, 102, 105], the Asp dyad can in principle exist in three protonation states, a deprotonated, a mono-protonated or a doubly protonated form. Because hydrogen atoms cffe invisible in the X-ray structure, evidence for a specific protonation state must be inferred indirectly by spectroscopic or titration measurements. Up to now, the existence of the doubly protonated, neutral form hfree enzyme. The existence of the deprotonated, doubly negative form is supported by a recent NMR study [102] at pH 6. However, this study has been subjected to criticism [106] and it is not conclusive. Our ab initio simulations of this form show that the Asp dyad is unstable even in the ps timesccde because of the strong Asp-Asp repulsion, which turns out to be -t-30 kcal/mol as estimated with a simple electrostatic model [100]). Thus, our calculations do not support the existence of this form. 

---