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Based on the molar absorptivities of the products, the glucose oxidase peroxidase assay may be expected to yield higher sensitivity and a lower detection limit. Ideally, the indicator enzyme converts primary product into measured product in a linear manner, meaning that every molecule of primary product is instantaneously converted, regardless of substrate concentration. To accomplish this, primary product concentrations are kept low, so that they fall into the linear region of the saturation kinetics curve. For linear conversion at all analyte (primary substrate) concentrations, the effective rate of the indicator reaction, (Veff)ind, must equal Vmax for the primary reaction: Vmax prim Veff ind Vmax ind =f1 Km;P1 = P1 Km;S2 = S2 g 3:11

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Nodes with many arrows coming into them are highly dependent on other services, and are deserving of extra redundancy engineering and aggressive monitoring. Spend the engineering effort and money to make these services reliable, as they re likely to affect many users or many other services. Routers introduce dependencies when there s only one path between services. If you find a router in the middle of a graph, remove it by adding a redundant routing device. Your monitoring tools may have network dependencies as well. Something as simple as netstat -r, which checks routing tables, uses NIS to look up hostnames for gateways and network names. Stick with the netstat -rn form, using numeric representation of IP addresses, to make netstat a standalone management tool. A dependency graph with cycles represents a network that cannot boot itself. During the process of starting up, you ll hit a race condition in which some service may be waiting on another one, which is waiting on some other host that forms a dependency loop. Consider a host that issues a route command during booting to find a network service on the far side of a router. The route utility uses a hostname, which is resolved via DNS. If your only authoritative DNS server is also on the far side of the router, there s no way that host is coming up all the way. Remember, after a major reboot, your DNS cache servers may not be useful. Just because it worked fine right up until the failure doesn t mean you can boot your way out of trouble. Break the cycles and make your network more resilient to massive failures or recovery efforts such as blackouts or disaster recovery processes.

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Naming and directory services are almost invariably at the top of the dependency charts, so we ll look at ways of hardening them and providing adequate capacity to prevent them from starting a Chernobyl-scale chain reaction on the network.

Since Km;P1 is a characteristic of the indicator enzyme, and [P1] is dictated by the analyte concentration, the only variables that can be controlled experimentally are Vmax ind , which is equal to kcat Eind (cf. 2) and the cosubstrate concentration [S2]. For this reason, a large excess (100-fold or more) of indicator enzyme is employed, in addition to saturating levels of cosubstrate (O-dianisidine in Eq. 3.8, and NADP in Eq. 3.10). When making initial rate measurements with coupled enzyme systems, there is often a signi cant lag time during which the linkage products build up to steadystate concentrations.6 Remember that some dehydrogenase reactions possess unfavorable equilibria; lactate dehydrogenase, for example, which catalyzes the reaction shown in Eq. 3.12: Lactate NAD Pyruvate NADH H 3:12

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prefers to convert pyruvate to lactate K 5 10 5 at pH 7. For this reason, coupling lactate dehydrogenase to a lactate-producing enzyme will not be effective, unless a trapping reagent is used to remove pyruvate and force the reaction towards products. For example, at pH 9.5, phenylhydrazine will trap pyruvate to produce a phenylhydrazone, shifting the lactate dehydrogenase equilibrium to K 2 10 2 .7 Alternatively, NADH may be trapped with an electron-transfer reagent such as phenazine methosulfate (PMS), which is colorless in the oxidized form but absorbs at 388 nm in the reduced form. 3.4. CLASSIFICATION OF METHODS Enzymatic assay methods are classi ed as xed-time assays, xed-change assays, or kinetic (initial rate) assays. Kinetic assays continuously monitor concentration as a function of time; pseudo- rst-order conditions generally apply up to $ 10% completion of the reaction to allow the initial reaction rate to be determined. If the initial substrate concentration is >10Km, then the initial rate is directly proportional to enzyme concentration. At low initial substrate concentrations (< 0:1 Km ), the initial rate will be directly proportional to initial substrate concentration (cf. 2). For enzyme quantitation, a plot of initial rate against [E] provides a linear