Reducing agents, enzyme activation

The effects of ozone appear to be cumulative for initial exposures followed by adaptation. Five of six subjects exposed to 0.5 ppm ozone 2 hours/day for 4 days showed cumulative effects of symptoms and lung function tests for the first 3 days, followed by a return to near control values on day 4." In animals exposure to 0.3-3 ppm for up to 1 hour permits the animals to withstand multilethal doses for months afterwards. However, repeated exposures impart protection from all forms of lung injury (e.g., susceptibility to infectious agents, enzyme activities, inflammation). Initial ozone exposure may act to reduce cell sensitivity and/or increase mucus thickness, factors which may modify the accessibility and action of the gas. It is not known how variations in the length, frequency, or magnitude of exposure modify the time course for tolerance. 

As an additional step, glutaraldehyde, a common cross-linking agent, can be included in the aqueous protein solution. The formation of covalent intra- or interenzyme cross linkages will help prevent leakage of enzyme from support and improve stability, but will also reduce the enzyme activity by increasing the resistance to pore diffusion. ... 

Fructose bisphosphate aldolase of animal muscle is a Class I aldolase, which forms a Schiff base or imme intermediate between the substrate (fructose-1,6-bisP or dihydroxyacetone-P) and a lysine amino group at the enzyme active site. The chemical evidence for this intermediate comes from studies with the aldolase and the reducing agent sodium borohydride, NaBH4. Incubation of fructose bisphosphate aldolase with dihydroxyacetone-P and NaBH4 inactivates the enzyme. Interestingly, no inactivation is observed if NaBH4 is added to the enzyme in the absence of substrate. 

The requirement for reduction prior to DNA alkylation and crosslinking was first demonstrated by Iyer and Szybalski in 1964 [29], and can be induced both by chemical reducing agents such as sodium dithionite and thiols in vitro and by various reductive enzymes such as DT-diaphorase (NAD(P)H-quinone oxidoreduc-tase) in vitro and in vivo [47]. Much work to characterize the mechanism of reductive activation and alkylation has been carried out, principally by the Tomasz and Kohn groups, and Figure 11.1 illustrates a generally accepted pathway for mitomycin C [16, 48-50] based on these experiments, which is very similar to the mechanism originally proposed by Iyer and Szybalski [29]. 

Most poly(HA) depolymerases are inhibited by reducing agents, e.g., dithio-erythritol (DTT), which indicates the presence of essential disulfide bonds, and by serine hydrolase inhibitors such as diisopropyl-fluoryl phosphate (DFP) or acylsulfonyl derivates. The latter compounds covalently bind to the active site serine of serine hydrolases and irreversibly inhibit enzyme activity [48]. 

A final consideration is to provide adequate desalting of the reduced antibody molecule from excess reducing agent. If even a small amount of a thiol-containing reductant remains, subsequent conjugation with a maleimide-activated enzyme will be inhibited. 

There is an irreversible enzymatic inactivation reaction, which occurs during the oxidation of the cyclizable and noncyclizable diphenols to oquinones. This inactivation process has been interpreted as being the result of a direct attack of an o-quinone on a nucleophilic residue (His) near the active enzyme center or of an attack of a copper-bound hydroxyl radical generated by the Cu(I)-peroxide complex. However, the latter hypothesis seems to be more probable, because inactivation also occurs in the presence of reducing agents that remove the o-quinones generated. 

At present the reasons for the differences in activity are not completely clear, but are discussed in Chapter 7. A practical consequence is that in order to estimate the amount of enzyme, it is necessary to activate the enzyme fully before assay. This can be done by incubating the enzyme for a sufficient length of time under hydrogen, or, more rapidly, with a strong reducing agent such as dithionite + methyl viologen. 

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