Determination of the catalytic site content of a polyclonal catalytic antibody preparation

1998 ◽  
Vol 26 (2) ◽  
pp. S170-S170 ◽  
Author(s):  
MARINA RESMINI ◽  
SHERAZ GUL ◽  
SANJIV SONKARIA ◽  
GERARD GALLACHER ◽  
KEITH BROCKLEHURST
Keyword(s):  
Catalytic Site ◽  
Catalytic Antibody ◽  
Antibody Preparation

scholarly journals Evidence for ‘lock and key’ character in an anti-phosphonate hydrolytic antibody catalytic site augmented by non-reaction centre recognition: variation in substrate selectivity between an anti-phosphonate antibody, an anti-phosphate antibody and two hydrolytic enzymes

2004 ◽  
Vol 381 (1) ◽  
pp. 125-130 ◽  
Author(s):  
Sanjiv SONKARIA ◽  
Guillaume BOUCHER ◽  
José FLÓREZ-ÁLVAREZ ◽  
Bilal SAID ◽  
Syeed HUSSAIN ◽  
...  
Keyword(s):  
Hydrolytic Enzymes ◽  
Catalytic Site ◽  
Catalytic Antibody ◽  
Substrate Selectivity ◽  
Reaction Centre ◽  
Antibody Preparation ◽  
Leaving Group ◽  
Catalytically Active ◽  
Group Part ◽  
Kinetic Selectivity

The substrate selectivities of an anti-phosphonate and an anti-phosphate kinetically homogeneous polyclonal catalytic antibody preparation and two hydrolytic enzymes were compared by using hapten-analogous and truncated carbonate and ester substrates each containing a 4-nitrophenolate leaving group. Syntheses of the truncated substrates devoid of recognition features in the non-leaving group parts of the substrates are reported. The relatively high kinetic selectivity of the more active anti-phosphonate antibody preparation is considered to depend on a relatively rigid catalytic site with substantial reaction centre specificity together with other important recognition interactions with the extended non-leaving group part of the substrate. In contrast, the less catalytically active, more flexible anti-phosphate antibody exhibits much lower kinetic selectivity for the substrate reaction centre comparable with that of the hydrolytic enzymes with activity much less dependent on recognition interactions with the non-leaving group part of the substrate. The ways in which haptenic flexibility and IgG architecture might contribute to the differential kinetic selectivities are indicated.

EVALUATION OF A KINETIC APPROACH TO THE DETERMINATION OF CATALYTIC SITE CONTENT IN ENZYMES AND ENZYME-LIKE CATALYSTS

1999 ◽  
Vol 27 (1) ◽  
pp. A37-A37
Author(s):  
SANJIV SONKARIA ◽  
SHERAZ GUL ◽  
MARINA RESMINI ◽  
KEITH BROCKLEHURST
Keyword(s):  
Catalytic Site ◽  
Kinetic Approach

scholarly journals Polyclonal antibody-catalysed amide hydrolysis

1992 ◽  
Vol 284 (3) ◽  
pp. 675-680 ◽  
Author(s):  
G Gallacher ◽  
M Searcey ◽  
C S Jackson ◽  
K Brocklehurst
Keyword(s):  
Polyclonal Antibody ◽  
Carboxy Group ◽  
Catalytic Antibody ◽  
Amino Group ◽  
Immunization Schedule ◽  
Keyhole Limpet Haemocyanin ◽  
Antibody Preparation ◽  
Amide Hydrolysis ◽  
Hydrolysis Of ◽  
Ester Substrate

1. The activated amide (4-nitroanilide), N-(4-nitrophenyl) N'-butyl-1,4-phenylenediacetamide (III) was synthesized. 2. A polyclonal antibody preparation (PCA 270-29) was elicited in a multigeneration cross-bred sheep (no. 270) and isolated 29 weeks into the immunization schedule by procedures described previously for PCA 270-22 [Gallacher, Jackson, Searcey, Badman, Goel, Topham, Mellor & Brocklehurst (1991) Biochem J. 271, 871-881]. These involved the use of an amide conjugate bonded through the carboxy group of 4-nitrophenyl 4′-carboxymethylphenyl phosphate and an amino group of keyhole-limpet haemocyanin as the immunogen. 3. PCA 270-29 was shown to catalyse the hydrolysis of both the carbonate ester substrate 4-nitrophenyl 4′-(3-aza-2-oxoheptyl)phenyl carbonate (I) and the amide substrate (III). Both catalyses obeyed the Michaelis-Menten equation with the following values of the parameters at 25 degrees C: for the hydrolysis of (I) at pH 8.0, Km = 3.96 +/- 0.28 microM and k(cat.) = 0.135 +/- 0.004 s-1 (k(non-cat.) = 1.99 x 10(-4) s-1); for the hydrolysis of (III) at pH 9.0, Km = 5.4 +/- 1.4 microM and k(cat.) = (5.95 +/- 0.75) x 10(-5) s-1 (k(non-cat.) = approx. 2 x 10(-7) s-1). 4. The finding that PCA 270-29 is almost equally effective as a catalyst for the hydrolysis of the amide (III) as for that of the carbonate ester (I) when allowance is made for the different intrinsic reactivities of the two types of substrate is discussed. The catalytic characteristics of PCA 270-29, the first example of a polyclonal catalytic antibody preparation shown to catalyse the hydrolysis of an amide and the first example of an antibody preparation (monoclonal or polyclonal) with such catalytic character to be produced by use of a phosphate immunogen, are compared with those of the small number of other antibody-mediated hydrolyses of amides in the literature.

scholarly journals A general kinetic approach to investigation of active-site availability in macromolecular catalysts

2000 ◽  
Vol 346 (1) ◽  
pp. 117-125 ◽  
Author(s):  
Marina RESMINI ◽  
Sheraz GUL ◽  
Steve CARTER ◽  
Sanjiv SONKARIA ◽  
Christopher M. TOPHAM ◽  
...  
Keyword(s):  
Active Site ◽  
Kinetic Method ◽  
Order Rate Constant ◽  
Catalytic Antibody ◽  
Binding Model ◽  
Antibody Preparation ◽  
Single Turnover ◽  
Step Model ◽  
Catalytic Material ◽  
Antibody Preparations

A potentially general kinetic method for the investigation of active-site availability in preparations of macromolecular catalysts was developed. Three kinetic models were considered: (a) the conventional two-step model of enzyme catalysis, where the preparation contains only active catalyst (Ea) and inert (i.e. non-binding, non-catalytic) material (Ei); (b) an extension of the conventional model (a) involving only Ea and Ei, but with non-productive binding to Ea (in addition to productive binding); (c) a model in which the preparation contains also binding but non-catalytic material (Eb), predicted to be present in polyclonal catalytic antibody preparations. The method involves comparing the parameters Vmax and Km obtained under catalytic conditions where substrate concentrations greatly exceed catalyst concentration with those (k, the limiting value of the first-order rate constant, kobs, at saturating concentrations of catalyst; and K) for single-turnover kinetics, in which the reverse situation obtains. The active-site contents of systems that adhere to model (a) or extensions that also lack Eb, such as the non-productive binding model (b), may be calculated using [Ea]T = Vmax/k. This was validated by showing that, for α-chymotrypsin, identical values of [Ea]T were obtained by the kinetic method using Suc-Ala-Ala-Pro-Phe-4-nitroanilide as substrate and the well-known ‘all-or-none’ spectroscopic assay using N-trans-cinnamoylimidazole as titrant. For systems that contain Eb, such as polyclonal catalytic antibody preparations, Vmax/k is more complex, but provides an upper limit to [Ea]T. Use of the kinetic method to investigate PCA 271-22, a polyclonal catalytic antibody preparation obtained from the antiserum of sheep 271 in week 22 of the immunization protocol, established that [Ea]T is less than approx. 8% of [IgG], and probably less than approx. 1% of [IgG].

Solution-phase immunoassay for determination of cortisol in serum by capillary electrophoresis

1995 ◽  
Vol 41 (9) ◽  
pp. 1403-1406 ◽  
Author(s):  
D Schmalzing ◽  
W Nashabeh ◽  
M Fuchs
Keyword(s):  
Capillary Electrophoresis ◽  
Serum Cortisol ◽  
Laser Induced Fluorescence ◽  
Solution Phase ◽  
Serum Samples ◽  
Rapid Separation ◽  
Antibody Preparation ◽  
Assay Performance ◽  
Assay Protocol

Abstract We describe a competitive solution-phase immunoassay for serum cortisol determination that involves capillary electrophoresis (CE) combined with laser-induced fluorescence for separation and quantification. A polyclonal antibody preparation and fluorescein-labeled cortisol are used as assay reagents. 8-Anilino-1-naphthalenesulfonic acid was added to the serum sample during the assay to promote the release of cortisol from endogenous binding proteins. Conditions for the rapid separation of free and bound labeled antigen by CE were developed. Aspects of assay performance are evaluated in this report. The resulting assay protocol allows for the analysis of serum samples without extraction or other sample preparation steps.

An immunochemical procedure for determination of mitochondrial aspartate aminotransferase in human serum.

1980 ◽  
Vol 26 (12) ◽  
pp. 1694-1700 ◽  
Author(s):  
R Rej
Keyword(s):  
Human Serum ◽  
Aspartate Aminotransferase ◽  
Room Temperature ◽  
Rabbit Antiserum ◽  
Complete Removal ◽  
Measurable Effect ◽  
Excellent Correlation ◽  
Antibody Preparation ◽  
Mitochondrial Aspartate Aminotransferase

Abstract An immunochemical procedure is described for quantitation of mitochondrial aspartate aminotransferase (m-AspAT; EC 2.6.1.1) activity in human serum specimens. Antibodies directed against purified soluble aspartate aminotransferase (s-AspAT) from human erythrocytes were produced in rabbits and partly purified. Antibody sufficient for analyses of > 6000 specimens could be obtained from 15 mL of rabbit antiserum; contaminant AspAT activity of the antibody preparation was < 0.4 U/L. Addition of antibody directly to purified AspAT isoenzymes resulted in inhibition of s-AspAT but had no measurable effect upon m-AspAT. Antibody is incubated with serum in the presence of polyethylene glycol for 60 min at room temperature, then 60 min at 4 degrees C, and centrifuged (7000 x g, 4 degrees C, 15 min). No detectable s-AspAT activity remains in the supernatant fluid; thus m-AspAT activity can be measured directly. Precision, both within-day and day-to-day, was < 1 U/L, or 3.0% of residual m-AspAT activity. The method completely removed 1200 U of purified s-AspAT activity per liter; addition of s-AspAT to serum in increasing concentrations of about 500 U/L had no effect upon the measurement of residual m-AspAT activity. Results of the procedure described showed excellent correlation with those by an alternative procedure involving antibodies directed against m-AspAT. Addition of both anti-s- and anti-m-AspAT antibodies resulted in complete removal of serum AspAT activity. Univalent Fab fragments prepared anti-s-AspAT antibodies were capable of directly inhibiting s-AspAT activity without precipitation. Although a homogeneous immunoinhibition assay was possible, the greater precision of the precipitation assay made it preferable.

Determination of the catalytic site of creatine kinase by site-directed mutagenesis

1994 ◽  
Vol 1206 (1) ◽  
pp. 97-104 ◽  
Author(s):  
M. Lijun Lin ◽  
Benjamin Perryman ◽  
David Friedman ◽  
Robert Roberts ◽  
Tony S. Ma
Keyword(s):  
Creatine Kinase ◽  
Catalytic Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Kinetic and Titration Methods for Determination of Active Site Contents of Enzyme and Catalytic Antibody Preparations

Methods ◽  
2001 ◽  
Vol 24 (2) ◽  
pp. 153-167 ◽  
Author(s):  
Keith Brocklehurst ◽  
Marina Resmini ◽  
Christopher M. Topham
Keyword(s):  
Active Site ◽  
Catalytic Antibody ◽  
Antibody Preparations

scholarly journals Evidence that the mechanism of antibody-catalysed hydrolysis of arylcarbamates can be determined by the structure of the immunogen used to elicit the catalytic antibody

2007 ◽  
Vol 401 (3) ◽  
pp. 721-726 ◽  
Author(s):  
Guillaume Boucher ◽  
Bilal Said ◽  
Elizabeth L. Ostler ◽  
Marina Resmini ◽  
Keith Brocklehurst ◽  
...  
Keyword(s):  
Phenyl Group ◽  
Catalytic Antibody ◽  
Antibody Preparation ◽  
Tetrahedral Intermediate ◽  
Rate Determining Step ◽  
Catalysed Reaction ◽  
Leaving Group ◽  
Antibody Catalysis ◽  
Elicitation Process ◽  
Hydrolysis Of

A kinetically homogeneous anti-phosphate catalytic antibody preparation was shown to catalyse the hydrolysis of a series of O-aryl N-methyl carbamates containing various substituents in the 4-position of the O-phenyl group. The specific nature of the antibody catalysis was demonstrated by the adherence of these reactions to the Michaelis–Menten equation, the complete inhibition by a hapten analogue, and the failure of the antibody to catalyse the hydrolysis of the 2-nitrophenyl analogue of the 4-nitrophenylcarbamate substrate. Hammett σ–ρ analysis suggests that both the non-catalysed and antibody-catalysed reactions proceed by mechanisms in which development of the aryloxyanion of the leaving group is well advanced in the transition state of the rate-determining step. This is probably the ElcB (elimination–addition) mechanism for the non-catalysed reaction, but for the antibody-catalysed reaction might be either ElcB or BAc2 (addition–elimination), in which the elimination of the aryloxy group from the tetrahedral intermediate has become rate-determining. This result provides evidence of the dominance of recognition of phenolate ion character in the phosphate hapten in the elicitation process, and is discussed in connection with data from the literature that suggest a BAc2 mechanism, with rate-determining formation of the tetrahedral intermediate for the hydrolysis of carbamate substrates catalysed by an antibody elicited by a phosphonamidate hapten in which phenolate anion character is minimized. The present paper contributes to the growing awareness that small differences in the structure of haptens can produce large differences in catalytic characteristics.

Sign in / Sign up

Export Citation Format

Share Document