Inactivation, dialysis

Elfriede Pistorius and I were not successful in repeating Chans experiments on dialysis inactivation with pure lipoxygenase-1. Using atomic absorption analysis we did find, however, that all four isoenzymes... 

If the inhibitor combines irreversibly with the enzyme—for example, by covalent attachment—the kinetic pattern seen is like that of noncompetitive inhibition, because the net effect is a loss of active enzyme. Usually, this type of inhibition can be distinguished from the noncompetitive, reversible inhibition case since the reaction of I with E (and/or ES) is not instantaneous. Instead, there is a time-dependent decrease in enzymatic activity as E + I El proceeds, and the rate of this inactivation can be followed. Also, unlike reversible inhibitions, dilution or dialysis of the enzyme inhibitor solution does not dissociate the El complex and restore enzyme activity. 

Mechanism-based inactivation results in formation of a covalent adduct between the active inhibitor and the enzyme, or between the active inhibitor and a substrate or cofactor molecule. If the mechanism involves covalent modification of the enzyme, then one should not be able to demonstrate a recovery of enzymatic activity after dialysis, gel filtration, ultrafiltration, or large dilution, as described in Chapters 5 to 7. Additionally, if the inactivation is covalent, denaturation of the enzyme should fail to release the inhibitory molecule into solution. If a radiolabeled version of the inactivator is available, one should be able to demonstrate irreversible association of radioactivity with the enzyme molecule even after denaturation and separation by gel filtration, and so on. In favorable cases one should likewise be able to demonstrate covalent association of the inhibitor with the enzyme by a combination of tryptic digestion and LC/MS methods. 

MHbR has been purified from erythrocytes of many species including man (H21, K8, Kll, K12, K14) and from brewers yeast (A6). The 1000-fold purified preparation obtained by Kiese (K14) has been shown, by means of chromatographical studies, to contain FAD. Thus the enzyme has been identified as a flavoprotein. However, the same author could not find any further activating effect of FAD or FMN on active preparations, nor a reactivation of the enzyme previously inactivated by dialysis (K14). 

Silverman has pointed out that several criteria must be met to demonstrate that a compound is a true suicide substrate 1101 (1) Loss of enzyme activity must be time-dependent, and it must be first-order in [inactivator] at low concentrations and zero-order at higher concentrations (saturation kinetics), (2) substrate must protect the enzyme from inactivation (by blocking the active site), (3) the enzyme must be irreversibly inactivated and be shown to have a 11 stoichiometry of suicide substrate active site (dialysis of enzyme previously treated with radiolabeled suicide substrate must not release radiolabel into the buffer), (4) the enzyme must unmask the suicide substrate s potent electrophile via a catalytic step,1121 and (5) the enzyme must not be covalently labeled with the activated form of the suicide substrate following its escape from the active site (the presence of bulky scavenging thiol nucleophiles in the buffer must not decrease the observed rate of inactivation). 

Pancreatic amylase is very labile and sensitive to its chemical environment. Its lability is accelerated by purification and by such factors as dilution of its aqueous solutions, dialysis of its aqueous solutions against water, unfavorable hydrogen ion activities and unfavorable temperatures.29-31, 36,33 The loss of amylase activity in solutions of pancreatic amylase increases with increasing temperature and is very rapid between 50° and 60°. The inactivation of pancreatic amylase in aqueous solution may be retarded by the addition of certain anions, of which the chloride ion is outstanding 37-39 by the addition of certain cations, of which... 

Similarly, the addition of substrate to hydrolysis mixtures that had reached stages of very slow rates of change with dialysis resulted in extensive hydrolysis of the added substrate. Comparisons showed that the new substrate was hydrolyzed to practically the same extent in the same time as the original substrate.41 These findings show not only that active amylase was present but that no appreciable inactivation of the amylase had taken place in the dialyzing hydrolysis mixtures when the... 

On the other hand, marked inactivation of the amylase (86 to 87 %) occurred when solutions of the same purified pancreatic amylase preparations were dialyzed under the same conditions but in the absence of substrate.41 These results give experimental evidence for the suggestion often advanced that the amylase unites with its substrate, in this case with the larger less readily dialyzable products of the hydrolysis of starch, and thus is protected from appreciable inactivation due to dialysis. 

Standard tests, dialysis, heat inactivation, and the effects of changing the pH on the catalytic activity of the preparations, were all consistent with the idea that enzymes had the properties ascribed to proteins. Between 1920 and 1930 increasing numbers of enzymes were isolated by Willstatter and partially purified. Assays for purification and the extent achieved were not sufficiently rigorous to exclude the possi-... 

Evidence that Epoxycreatine is an Affinity Label. All the available evidence is consistent with the hypothesis that epoxycreatine behaves like an affinity label for the enzyme and that it is attacked by a carboxylate group of the enzyme. That is, it inactivated the enzyme rapidly at 0°C. Inactivation was complete and activity did not return upon exhaustive dialysis. Creatine was shown to give protection against the inactivation in the expected manner. Most importantly, though, the irreversible binding of the Inhibitor was shown to be stoichiometric using [ Cj-epoxycrea-tine that is, one and only one inhibitor molecule becomes bound per active site, even in the presence of excess inhibitor. 

Walters and Loring (88) have purified a 3 -nucleotidase about 50-fold from mung bean sprouts (Phaseolus aureus Roxb.). The enzyme hydrolyzes 3 -AMP, 3 -GMP, 3 -CMP in decreasing order and also hydrolyzes the 3 -phosphate group of coenzyme A. (89), but it has no significant activity for 2 - or 5 -ribonucleotides. For 3 -GMP, 3 -AMP, 3 -UMP, and 3 -CMP, Km values are 0.67, 1.1, 7.7, and 15 mM, respectively. The enzyme preparation also contained acid stable ribonuclease activity (89). Both 3 -nucleotidase and acid ribonuclease were inactivated reversibly at pH 5.0 and by dialysis and this inactivation could be prevented by Zn2+. The two activities were similarly inactivated by heat at pH 5 and 7.5. Such data indicate that the two are metalloproteins— probably zinc metalloproteins. These similarities and other kinetic data provide evidence that the 3 -nucleotidase and ribonuclease activities reside in the same protein. 

MacIntyre and Dean (119) report that acid phosphatase from D. melanogaster has slow and fast electrophoretic variants specified by co-dominant alleles. Thus, acid phosphatases AA, BB, and AB were studied. Types AA and BB could be inactivated by exposure to acid. Reactivation of enzymic activity could be accomplished by dialysis against buffers at pH 6.5. Mixtures of AA and BB produced some AB reconstituted enzyme. From this evidence it seems very probable that acid phosphatase, at least in this species, consists of at least two polypeptide chains. 

Preservatives such as sodium benzoate, sorbic acid, and methyl and propyl parabens have been used in liquid and semisolid dosage forms. There have been reports that the parabens have been inactivated when used in the presence of various surfactants. This loss of activity was thought to be due to the formation of complexes between the preservative and the surfactant. The interaction between polysorbate (Tween) 80 and the parabens has been demonstrated by a dialysis technique (Ravin and Radebaugh, 1990). It has also been shown that molecular complexes form when the parabens are mixed with polyethylene glycol (PEG) and methylcellulose. The degree of binding was less than that observed with Tween 80. Sorbic acid also interacts with Tweens but does not interact with PEGs. The quaternary ammonium compounds are also bound by Tween 80, which reduces their preservative activity. 

---