Enzyme sensitive units

Incorporation of enzyme-sensitive units in the polymer side chain can be done either after the main polymer has been synthesised or the enzyme-sensitive functionality can be present in the monomer before polymerisation (Fig. 6.10). Attachment to the polymer is typically done via standard SPPS protocols. Both peptides and DNA fragments have been attached to polymers obtained through ring opening polymerisation of norbornene using either N-hydroxysuccinimide (NHS) activation or A, A -tetramethyl-... 

Serum retains its creatine kinase activity at —25°C for at least a month without change (H7). In the colorimetric procedure (H7), which is especially sensitive, units of creatine kinase activity are expressed as pmoles creatine/hour/ml serum at 37°C, with a normal range of 0.6-4.0 units per ml for men and 0.6-2.6 units per ml for women (H9), thus clearly establishing a sex difference not apparent in the other semm enzymes so far considered. 

In many situations, the actual molar amount of the enzyme is not known. However, its amount can be expressed in terms of the activity observed. The International Commission on Enzymes defines One International Unit of enzyme as the amount that catalyzes the formation of one micromole of product in one minute. (Because enzymes are very sensitive to factors such as pH, temperature, and ionic strength, the conditions of assay must be specified.) Another definition for units of enzyme activity is the katal. One katal is that amount of enzyme catalyzing the conversion of one mole of substrate to product in one second. Thus, one katal equals 6X10 international units. 

Consistent with the two-electron donor nature of H2, the reaction behaved as an n=2 Nernst redox reaction. It showed a pH dependence of 66mV per pH unit, so again one proton was taken up for each electron. It is not known where all incoming protons are localized in the enzyme. The reaction shows that in addition to the light-sensitive hydrogen species bound to the active site in the Nia-C " state, a second hydrogen can react at the active site and deliver its two electrons to the enzyme. We hence proposed that the active site of the A. vinosum enzyme has two sites where hydrogen can bind. If H2 is completely removed, the Nia-C state persists for hours this is unlike the situation in redox titrations in the presence of redox mediators. As the active site in the Nig-SR state has one electron more than that in the Nia-C state, an Fe-S cluster has to be involved in this reaction with H2. 

Several things may be done if the researcher has difficulty in detecting an enzyme activity of interest in a homogenate, or elsewhere. A more sensitive assay technique may be used, if one is available. The concentration of enzyme may be increased, as the rate of product formation is directly proportional to [E]. The incubation time for enzyme with substrate can be increased, although the caveats discussed in O Section 3.3.2 must be borne in mind. The reaction volume may also be increased, while maintaining concentrations of reactants constant this approach is particularly useful if product is separated and detected by chromatography, or if a column is used to separate radiolabeled substrate from product, because the increased amount of product formed in unit time will result in enhanced signal size. 

Variations on the ABC technique can also be used to incorporate different enzymes that result in different chromogenic products. Alkaline phosphatase ABC is one example. The difficulty with this system is the consumption of endogenous alkaline phosphatase, which is more prevalent than peroxidase and is harder to remove. However, the alkaline phosphatase enzyme does provide more product per unit than does peroxidase and is therefore a slightly more sensitive means of detection. Endogenous alkaline phosphatase can be blocked by an incubation in 3 mMlevamisole for 15 min, but some enzyme may escape consumption. Alkaline phosphatase has many substrates, too, the most popular being BCIP/NBT, which precipitates to a dark blue (see Chapter 25). 

More recently, Pearce and Heydeman suggested non-oxidative removal of ethylene glycol units as acetaldehyde by a membrane-bound, oxygen-sensitive enzyme of a novel type, i.e., diethylene glycol lyase (18). Schoberl suggested that PEG was catabolized by Ci step, liberating formate which was metabolized by a serine pathway (19). 

The ATP synthase (EC3.6.1.34, complex V) that transports H"" is a complex molecular machine. The enzyme consists of two parts—a proton channel (Fq, for oligomycin-sensitive ) that is integrated into the membrane and a catalytic unit (Fi) that protrudes into the matrix. The Fo part consists of 12 membrane-spanning c-peptides and one a-subunit. The head of the Fi part is composed of three a and three p subunits, between which there are three active centers. The stem between Fo and Fi consists of one y and one e subunit. Two more polypeptides, b and 8, form a kind of stator, fixing the a and p subunits relative to the Fo part. 

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