Enzyme unconjugated

Purify the conjugate from excess reactants by dialysis or gel filtration using a desalting resin. Use 0.01 M sodium phosphate, 0.15M NaCl, pH 7.0, as the buffer for either operation. The conjugate may be further purified by removal of unconjugated enzyme using one of the methods described in Section 1.5, this chapter. 

Removal of Unconjugated Enzyme from Antibody-Enzyme Conjugates... 

Continue to wash the gel with 0.15M NaCl (saline solution) until the absorbance at 280nm is down to baseline. The protein eluting from the column at this point is unconjugated enzyme. 

Serum ALP and total bilirubin (unconjugated and conjugated fractions) are traditionally used to monitor cholestatic injury. The ALP families of enzymes are zinc metalloproteases that are present in nearly all tissues. In the liver, ALP is immu-nolocalized to the microvili of the bile canaliculus [124]. Increased synthesis of ALP and its release into the circulation occurs within hours of cholestatic injury [129]. Serum assays of 5 -nucleotidase (5 -NT) or y-glutamyltransferase activity (GGT) are used to confirm the liver as the specific origin for the elevation of ALP. Increases in serum bilirubin or bile acids are usually the result of bile retention subsequent to impaired bile flow, increased production associated with accelerated erythrocyte destruction, or altered bilirubin metabolism [129]. 

Most glucocorticosteroid are metabolized in the liver to hydroxy- and ketosteroid metabolites which are excreted by the kidneys as glucuronides, sulfates and unconjugated products. Enzyme-inducing agents will diminish the efficacy of glucocorticosteroids. 

Oxalocrotonate tautomerase. This bacterial enzyme, which functions in the degradation of toluene (Chapter 25), is actually an isomerase. It catalyzes rapid interconversion of an unconjugated unsaturated a-oxoacid such as 4-oxalocrotonate with an intermediate enol (which may leave the enzyme) and the isomeric conjugated oxoacid (Eq. 13-31).168-170 A related 5-carboxymethyl-2-hydroxymuconate isomerase... 

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