Enzyme elution

Reverse transcriptase (from avian or murine RNA tumour viruses) [9068-38-6] [EC 2.7.7.49]. Purified by solubilising the virus with non-ionic detergent. Lysed virions were adsorbed on DEAE-cellulose or DEAE-Sephadex columns and the enzyme eluted with a salt gradient, then chromatographed on a phosphocellulose column and enzyme activity eluted in a salt gradient. Purified from other viral proteins by affinity chromatography on a pyran-Sepharose column. [Verna Biochim Biophys Acta 473 1 7977 Smith Methods Enzymol 65 560 1980 see commercial catalogues for other transcriptases.]... 

Figure 7. HPLC kinetic profiles of reaction products of pectate lyases obtained from chromatofocused fractions of Erwinia chrysanthemi culture supernatants. Chromatofocused enzymes, eluted at pH 8.6 (A), pH 8.3 (B), pH 6.0 (C), and < pH 6.0 (D), were assayed under conditions similar to those described in Figure 5.

Ammonium sulfate concentration at which the enzymes eluted from various solid supports. From Mevarech et al. (1976), with permission. 

Under the proper conditions, all enzyme activity is bound to the column. The apparent activity of the immobilized enzyme relative to assays done in free solution is in the range of 80-90%. The enzyme can be eluted in active form (about 95% of the initial activity) with 25% glycerol. The matrix is reusable after the enzyme elution. 

The dialyzed enzyme solution was now subjected to a repetition of the preceding procedures admixture of suflBcient calcium phosphate gel to adsorb protein but leave the enzyme in solution centrifugation and addition of more gel to the supernatant to adsorb the enzyme elution of the enzyme from the gel with a mixture of 0.15 M acetate and 0.015 M citrate at pH 4.5 addition of solid ammonium sulfate to the eluate to 55% saturation and precipitation of the enzyme. At this stage, the purifications ranged from 650- to 1100-fold with a recovery of approximately 20-30% of the activity present in the crude red cell hemolysate. Solution of this precipitate, dialysis treatment with solid ammonium sulfate and collection of the precipitate appearing between 40 and 55% saturation yielded a preparation that represented a 1500-fold purification. The preparations were stable when left sedimented in the ammonium sulfate solution. 

Non-specific enzyme elution procedures may however gradually inactivate and decrease the utility of antibody supports. For instance, exposure of antibody supports to pH below 4.0 may decrease the affinity for the antigen and promote susceptibility to proteolysis [52]. Improvement in resistance to proteolysis has however been achieved by controlled modifications of matrix associated antibody with polyethylene glycol [85]. 

On the basis of the elution volumes of the enzyme eluted from the column with an immobilized inhibitor, using the solutions of various concentrations of a soluble inhibitor, we can determine the inhibition constants both with the immobilized inhibitor and with the soluble inhibitor employed [26,27]. Using the same inhibitor for the immobilization and elution, we can draw direct conclusions about the effects... 

N-(l-pyrenil)maleimide <15> (<15> complete loss of catalytic activity, but modified enzyme is able to bind jS-D-fructose 6-phosphate, the presence of MgATP " completely protects the enzyme activity, the modified enzyme elutes as a monomer [67]) [67]... 

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