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A polyclonal antibody preparation with Michaelian catalytic properties.

ABSTRACT: 1. 4-Nitrophenyl 4'-(3-aza-2-oxoheptyl)phenyl carbonate (I), an amide conjugate (XI) involving the carboxy group of 4-nitrophenyl 4'-carboxymethylphenyl phosphate and an amino group of keyhole-limpet haemocyanin, and a fluorescein derivative (XVII) were synthesized. 2. The conjugate (XI) was used as an immunogen with which to raise polyclonal antibodies in multigeneration cross-bred sheep; the fluorescent derivative (XVII) was used for the initial assessment of the antisera via binding assays monitored by fluorescence polarization; the carbonate ester (I) was used as a chromogenic substrate for the investigation of catalytic activity. 3. The IgG from the antiserum of sheep no. 270 was isolated by Na2SO4 precipitation and chromatography on Protein G-Sepharose. 4. This preparation of IgG catalysed the hydrolysis of the carbonate ester (I); the catalysis at pH 8.0 and 25 degrees C obeyed Michaelis-Menten kinetics with at least 25 turnovers, Km = 3.34 microM, and lower limits for kcat. of 0.029 s-1 and for kcat./Km of 8.77 x 10(3) M-1.S-1, on the unlikely assumption that the concentration of catalytic antibody is provided by twice the total IgG concentration (two sites per molecule); probable estimates of the fraction of the total IgG that is anti-haptenic IgG and of the fraction of this that is catalytically active suggest that the values of kcat./Km are actually very much larger than these lower limits. 5. The failure of the antibody preparation to catalyse the hydrolysis of the isomeric 2-nitrophenyl carbonate (II), which differs from compound (I) only in the position of the nitro substituent in the leaving group, compels the view that catalytic activity is due to antibody rather than contaminant enzyme; this conclusion is supported by (a) the failure of the following to discriminate effectively between the isomeric substrates (I) and (II): pig liver carboxylesterase, rabbit liver carboxylesterase (collectively EC 3.1.1.1), whole serum from a non-immunized sheep and whole serum from a sheep immunized with a derivative of 3-O-methylnoradrenaline and (b) the lack of catalytic activity in IgG preparations from sheep immunized with sulphoxide or sulphone analogues of immunogen (XI). 6. The various parameters used for the comparison of the kinetic characteristics of hydrolytic catalytic antibodies are discussed. 7. The characteristics of hydrolysis of compound (I) catalysed by the present polyclonal antibody preparation are shown to be substantially better in most respects than those of analogous reactions of two other carbonate esters catalysed by monoclonal antibodies.

SUBMITTER: Gallacher G

PROVIDER: S-EPMC1151527 | biostudies-other | 1991 Nov

REPOSITORIES: biostudies-other

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Project description:We have performed a systematic variability study of polyclonal antibody catalysis by using five rabbits immunized with the same hapten. Important results from this work are the following. (1) Similarities were observed in the catalytic polyclonal antibodies derived from all five rabbits. Four of the five rabbits produced polyclonal samples that were nearly the same in terms of catalytic activity, whereas the fifth rabbit, designated as rabbit 2, displayed a somewhat higher level of catalytic activity. The catalytic activities (as kcat/kuncat) of these polyclonal samples were similar to that from the best murine monoclonal antibody that had been previously elicited by the same hapten. (2) Titre was not an accurate indicator of polyclonal antibody catalytic activity. (3) A mathematical analysis to describe a distribution of Michaelis-Menten catalysts was performed to help interpret our results. (4) Kinetic analysis indicated that the binding parameters of the different samples were remarkably homogeneous, because one or two components were all that were required to fit the on-rate and off-rate data satisfactorily. Interestingly, the most active catalytic polyclonal sample, that from rabbit 2, displayed the slowest off-rate (so slow it could not be measured) and thus the highest overall affinity. (5) Catalytic analysis of eluted fractions of antibody from a substrate column indicated that each polyclonal sample was also relatively homogeneous in terms of catalytic parameters. The main conclusion of our study is that for this hapten-animal system, the overall catalytic immune response is relatively consistent at two levels. Consistent catalytic activity was observed between the polyclonal samples elicited in the different animals, and the elicited hapten-specific polyclonal antibodies were relatively homogeneous in terms of binding and catalytic parameters within each immunized animal. The observed similarities of the catalytic activity in the different animals is surprising, because the immune response is based on specific binding of antibodies to hapten. There is no known selective pressure to maintain consistent levels of catalytic activity. Our results can therefore be interpreted as providing evidence that for this hapten there is a fixed relationship between hapten structure and catalytic activity and/or consistent genetic factors that dominate the catalytic immune response.

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A polyclonal antibody preparation with Michaelian catalytic properties.

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