Catalytic IgGs Research Articles

DNA-hydrolyzing catalytic IgGs from schizophrenia patients do not affect cell viability of the SH-SY5Y human neuroblastoma cell line

IntroductionDNA-hydrolyzing catalytic IgGs have caspase-dependent cytotoxic effects in autoimmune diseases. Recently, DNA-hydrolyzing IgGs have been discovered in schizophrenia. However, their cytotoxic properties have not been studied.ObjectivesTo assess the effect of serum IgGs with DNA-hydrolyzing activity of schizophrenia patients on the cell viability of the SH-SY5Y human neuroblastoma cell line.MethodsSerum of 8 patients with paranoid schizophrenia in the acute phase and 7 mentally and somatically healthy persons were used. IgG was purified from serum by affinity chromatography on Protein-G-Sepharose columns. The DNA hydrolyzing activity of IgG was assessed by the degree of hydrolysis of the pBluescript plasmid. The cell viability of the SH-SY5Y human neuroblastoma cell line after exposure to purified IgG preparations was assessed by high-throughput screening on the CellInsight CX7 platform (Thermo Scientific, USA) using the fluorescent dyes propidium iodide and Hoechst.ResultsOf the 8 IgG preparation obtained, 4 drugs had high DNA-hydrolyzing activity. All tested IgG preparations from healthy donors were inactive. One-way ANOVA analysis of the proportion of dead cells of the SH-SY5Y line after exposure to antibodies (0.1 mg/ml) showed no significant differences in the proportion of dead cells (p=0.688 after 24 hours; p=0.831 after 48 hours). Similar results were obtained at a higher concentration of antibodies - 0.2 mg/ml.ConclusionsThus, it has been shown in vitro that IgGs isolated from the serum of schizophrenia patients with or without DNA-hydrolyzing activity does not exhibit cytotoxic properties against the SH-SY5Y human neuroblastoma cell line. Support by Grant of RSF № 18-15-00053P.DisclosureNo significant relationships.

Natural Catalytic IgGs Hydrolyzing Histones in Schizophrenia: Are They the Link between Humoral Immunity and Inflammation?

Schizophrenia is known to be accompanied not only with an imbalance in the neurotransmitter systems but also with immune system dysregulation and chronic low-grade inflammation. Extracellular histones and nucleosomes as damage-associated molecular patterns (DAMPs) trigger systemic inflammatory and toxic reactions by activating Toll-like receptors. In this work, we obtained the first evidence that polyclonal IgGs of patients with schizophrenia effectively hydrolyze five histones (H1, H2a, H2b, H3, and H4). Several strict criteria were used to demonstrate that histone-hydrolyzing activity is a property of the analyzed IgGs. The IgGs histone-hydrolyzing activity level, depending on the type of histone (H1–H4), was statistically significantly 6.1–20.2 times higher than that of conditionally healthy donors. The investigated biochemical properties (pH and metal ion dependences, kinetic characteristics) of these natural catalytic IgGs differed markedly from canonical proteases. It was previously established that the generation of natural catalytic antibodies is an early and clear sign of impaired humoral immunity. One cannot, however, exclude that histone-hydrolyzing antibodies may play a positive role in schizophrenia pathogenesis because histone removal from circulation or the inflamed area minimizes the inflammatory responses. Thus, it can be assumed that histone-hydrolyzing antibodies are a link between humoral immunity and inflammatory responses in schizophrenia.

Hydrolysis by catalytic IgGs of microRNA specific for patients with schizophrenia.

Significant importance of autoimmune changes in the pathogenesis of schizophrenia (SCZ) is not established. Here, we present the first evidence that autoantibodies of 100% SCZ patients possess RNase activity: сCMP > poly(C) > poly(A) > yeast RNA. In addition, we have got an unexpected result: there was revealed site-specific hydrolysis of four known SCZ specific microRNAs (miR-137, miR-9-5p, miR-219-2-3p, and miR-219a-5p) playing an important role in the regulation of several genes functioning. Three major of cleavage sites are located in the microRNA loops or duplex parts directly articulated with the loops. RNase abzymes can contribute to decreasing of microRNAs effects on the functioning of numerous genes and the products of their transcription. Therefore, abzymes with RNase activity may be to some extent important for the development of schizophrenia. © 2018 IUBMB Life, 70(2):153-164, 2018.

Changes in haematopoietic progenitor colony differentiation and proliferation and the production of different abzymes in EAE mice treated with DNA.

Immunization of experimental autoimmune encephalomyelitis (EAE)‐prone C57BL/6 mice with MOG 35‐55 (a model used to study aspects of human multiple sclerosis) is known to lead to the production of various abzymes. The production of catalytic IgGs that can efficiently hydrolyse myelin basic protein (MBP), MOG and DNA is associated with changes in the profile of differentiation and level of proliferation of mice bone marrow haematopoietic stem cells (HSCs). As MOG simulates the production of abzymes with high DNase activity, we compared the effects of DNA and MOG immunization on EAE‐prone mice. In contrast to MOG, immunization with DNA leads to a suppression of proteinuria, a decrease in the concentrations of antibodies to MOG and DNA and a reduction in abzyme production. Immunization with DNA only resulted in a significant increase in DNase activity over 40 days where it became 122‐fold higher than before immunization, and fivefold higher when comparing to the maximal activity obtained after MOG treatment. DNA and MOG immunization had different effects on the differentiation profiles of HSCs, lymphocyte proliferation, and the level of apoptosis in bone marrow and other organs of mice. The data indicate that for C57BL/6 mice, DNA may have antagonistic effects with respect to MOG immunization. The usually fast immune response following MOG injection in C57BL/6 mice is strongly delayed after immunization with DNA, which is probably due to a rearrangement of the immune system following the response to DNA.

Generation of Catalytic Antibodies Is an Intrinsic Property of an Individual's Immune System: A Study on a Large Cohort of Renal Transplant Patients.

Renal transplant is the treatment of choice for patients with terminal end-stage renal disease. We have previously identified low levels of catalytic IgG as a potential prognosis marker for chronic allograft rejection. The origin and physiopathological relevance of catalytic Abs is not well understood, owing to the fact that catalytic Abs have been studied in relatively small cohorts of patients with rare diseases and/or without systematic follow-up. In the current study, we have followed the evolution of the levels of catalytic IgG in a large cohort of renal transplant patients over a 2-y period. Our results demonstrate that, prior to transplant, patients with renal failure present with heterogeneous levels of IgG hydrolyzing the generic proline-phenylalanine-arginine-methylcoumarinamide (PFR-MCA) substrate. PFR-MCA hydrolysis was greater for patients' IgG than for a therapeutic preparation of pooled IgG from healthy donors. Renal transplant was marked by a drastic decrease in levels of catalytic IgG over 3 mo followed by a steady increase during the next 21 mo. Patients who displayed high levels of catalytic IgG pretransplant recovered high levels of catalytic Abs 2 y posttransplant. Interestingly, IgG-mediated hydrolysis of a model protein substrate, procoagulant factor VIII, did not correlate with that of PFR-MCA prior transplantation, whereas it did 12 mo posttransplant. Taken together, our results suggest that the level of circulating catalytic IgG under pathological conditions is an intrinsic property of each individual's immune system and that recovery of pretransplant levels of catalytic IgG is accompanied by changes in the repertoire of target Ags.

Generation of catalytic antibodies is an intrinsic property of an individual’s immune system: a study on a large cohort of renal transplant patients

Abstract The origin and physiopathological relevance of catalytic antibodies is not well understood owing to the fact that catalytic antibodies have been studied in relatively small cohorts of patients with rare diseases and/or without systematic follow-up. In the present study, we have followed the evolution of the levels of catalytic IgG in a large cohort of 100 renal transplant patients over a 2-year period. Prior to transplant, hydrolysis of the generic substrate PFR-MCA was greater for patients’ IgG than for a therapeutic preparation of pooled IgG from healthy donors (6.6±0.9 vs 0.65±0.03 fmol/min per pmol). Renal transplant was marked by a drastic decrease in levels of catalytic IgG over 3 months (6.6±0.9 vs 2.4±0.2 fmol/min/pmol; P<0.0001) followed by a steady increase at 12 months (3.2±0.3 fmol/min/pmol; P=0.015) and further at 24 months (5.1±0.6 fmol/min/pmol; P=0.004). When divided into quartiles based on the rates of IgG-mediated PFR-MCA hydrolysis measured in pre-transplant samples, the IgG catalytic activity in the upper quartile of patients was significantly high both pre-transplant (12.03±1.6 vs 2.7±0.2 fmol/min/pmol, P<0.0001) and 24 months post-transplant (6.8±1.2 vs 4.6±0.7, fmol/min/pmol, P=0.0004). Interestingly, IgG-mediated hydrolysis of a model protein substrate, pro-coagulant factor VIII, did not correlate with that of PFR-MCA prior transplantation, while it did 12 months post-transplant (P<0.0001, R2=0.4). Taken together, our results suggest that the level of circulating catalytic IgG under pathological conditions is an intrinsic property of each individual’s immune system, and that recovery of pre-transplant levels of catalytic IgG is accompanied by changes in the repertoire of target antigens.

Why specific anti-integrase antibodies from HIV-infected patients can efficiently hydrolyze 21-mer oligopeptide corresponding to antigenic determinant of human myelin basic protein

Human immunodeficiency virus-infected patients possess anti-integrase (IN) catalytic IgGs and IgMs (abzymes), which, unlike canonical proteases, specifically hydrolyze only intact globular IN. Anti-myelin MBP abzymes from patients with multiple sclerosis and systemic lupus erythematosus efficiently hydrolyze only intact MBP. Anti-MBP and anti-IN abzymes do not hydrolyze several other tested control globular proteins. Here, we show that anti-IN abzymes efficiently hydrolyze a 21-mer oligopeptide (OP21) corresponding to one antigenic determinant (AGD) of MBP, whereas anti-MBP abzymes extremely poorly cleave oligopeptides corresponding to AGDs of IN. All sites of IgG-mediated and IgM-mediated proteolysis of OP21 by anti-IN abzymes were found for the first time by a combination of reverse phase and thin layer chromatography and mass spectrometry. Several clustered sites of OP21 cleavage were revealed and compared with the cleavage sites within the complete IN. Several fragments of OP21 had good homology with many fragments of the IN sequence. The active sites of anti-IN abzymes are known to be located on their light chains, whereas heavy chains are responsible for the affinity for protein substrates. Interactions of intact IN with both light and heavy chains of the abzymes provide high affinity for IN and the specificity of its hydrolysis. Our data suggest that OP21 interacts mainly with the light chains of polyclonal anti-IN abzymes, which possess lower affinity and specificity for substrate. The hydrolysis of the non-cognate OP21 oligopeptide may be also less specific than the hydrolysis of the globular IN because in contrast to previously described serine protease-like abzymes against different proteins, anti-IN abzymes possess serine, thiol, acidic, and metal-dependent protease activities.

IVIg Treatment Reduces Catalytic Antibody Titers of Renal Transplanted Patients

Catalytic antibodies are immunoglobulins endowed with enzymatic activity. Catalytic IgG has been reported in several human autoimmune and inflammatory diseases. In particular, low levels of catalytic IgG have been proposed as a prognostic marker for chronic allograft rejection in patients undergoing kidney transplant. Kidney allograft is a treatment of choice for patients with end-stage renal failure. Intravenous immunoglobulins, a therapeutic pool of human IgG, is used in patients with donor-specific antibodies, alone or in conjunction with other immunosuppressive treatments, to desensitize the patients and prevent the development of acute graft rejection. Here, we followed for a period of 24 months the levels of catalytic IgG towards the synthetic peptide Pro-Phe-Arg-methylcoumarinimide in a large cohort of patients undergoing kidney transplantation. Twenty-four percent of the patients received IVIg at the time of transplantation. Our results demonstrate a marked reduction in levels of catalytic antibodies in all patients three months following kidney transplant. The decrease was significantly pronounced in patients receiving adjunct IVIg therapy. The results suggests that prevention of acute graft rejection using intravenous immunoglobulins induces a transient reduction in the levels of catalytic IgG, thus potentially jeopardizing the use of levels of catalytic antibodies as a prognosis marker for chronic allograft nephropathy.

IVIg treatment reduces catalytic antibody titers of renal transplanted patients (P2208)

Abstract Catalytic antibodies are immunoglobulins endowed with enzymatic activity. Catalytic IgG has been reported in several human autoimmune and inflammatory diseases. In particular, low levels of catalytic IgG have been proposed as a prognostic marker for chronic allograft rejection in patients undergoing kidney transplant. Kidney allograft is treatment of choice for patients with end-stage renal failure. Intravenous immunoglobulins, a therapeutic pool of normal human IgG, is used in patients with donor-specific antibodies, alone or in conjunction with other immunosuppressive treatments, in order to desensitize the patients and prevent the development of acute graft rejection. In the present study, we followed for a period of 24 months the levels of catalytic IgG in a large cohort of patients undergoing kidney transplantation. Twenty-four percent of the patients received IVIg at the time of transplantation. Our results demonstrate a marked reduction in the levels of catalytic antibodies in all patients three months following kidney transplant. The decrease was significantly more pronounced in patients receiving adjunct IVIg therapy. The results suggests that prevention of acute graft rejection using intravenous immunoglobulins induces a transient reduction in the levels of catalytic IgG, thus potentially jeopardizing the use of levels of catalytic antibodies as a prognosis marker for chronic allograft nephropathy.

Immunization of rabbits with DNase II leads to formation of polyclonal antibodies with DNase and RNase activities

The serum of patients with many autoimmune (AI) diseases contains small fractions of antibodies possessing both DNase and RNase activities. It was shown that immunization of rabbits with DNA, RNA, DNase I and RNase leads to production of antibodies with DNase and RNase activities. It is not known whether anti-idiotypic antibodies against DNase II can possess DNase or RNase activity. Electrophoretically and immunologically homogeneous polyclonal IgGs (pIgGs) from the sera of rabbits immunized with DNase II were obtained by sequential chromatography of the serum proteins on Protein A-Sepharose and gel filtration. It was shown for the first time that immunization of healthy rabbits with bovine DNase II produces IgGs with intrinsic DNase and RNase activities. IgGs from rabbits immunized with BSA or non-immunized animals were catalytically inactive. It was shown that approximately 10% of the total IgG DNase and RNase activities belong to anti-idiotypic antibodies to DNase II ( approximately 0.1% of total pIgGs), while 90% of the activities did not interact with Sepharose bearing antibodies against DNase II and might be antibodies to nucleic acids bound to DNase II. Affinity chromatography on DNA-cellulose using elution of antibodies with different concentration of NaCl and an acidic buffer separated catalytic IgGs into five antibody subfractions, three of which hydrolyzed RNA faster than DNA while two subfractions demonstrated only DNase activity. Our data suggest that a fraction of abzymes from AI patients hydrolyzing both DNA and RNA can contain subfraction of antibodies against DNase II.

Antibodies against RNA hydrolyze RNA and DNA

Immunization of animals with DNA leads to the production of anti-DNA antibodies (Abs) demonstrating both DNase and RNase activities. It is currently not known whether anti-RNA Abs can possess nuclease activities. In an attempt to address this question, we have shown that immunization of three rabbits with complex of RNA with methylated BSA (mBSA) stimulates production of IgGs with RNase and DNase activities belonging to IgGs, while polyclonal Abs from three non-immunized rabbits and three animals immunized with mBSA are catalytically inactive. Affinity chromatography of IgGs from the sera of autoimmune (AI) patients on DNA-cellulose usually demonstrates a number of fractions, all of which effectively hydrolyze both DNA and RNA, while rabbit catalytic IgGs were separated into Ab subfractions, some of which demonstrated only DNase activity, while others hydrolyzed RNA faster than DNA. The enzymic properties of the RNase and DNase IgGs from rabbits immunized with RNA distinguish them from all known canonical RNases and DNases and DNA- and RNA-hydrolyzing abzymes (Abzs) from patients with different AI diseases. In contrast to RNases and AI RNA-hydrolyzing Abs, rabbit RNase IgGs catalyze only the first step of the hydrolysis reaction but cannot hydrolyze the formed terminal 2',3'-cyclophosphate. The data indicate that Abzs of AI patients hydrolyzing nucleic acids in part may be Abs against RNA and its complexes with proteins.

Antibodies against pancreatic ribonuclease A hydrolyze RNA and DNA

The sera of patients with autoimmune (AI) diseases contain antibodies with DNase and RNase activities. We have shown for the first time that immunization of healthy rabbits with RNase A conjugated with BSA produces a better immune response than immunization with pure RNase and induced IgGs with RNase and DNase activities, which were intrinsic properties of IgGs, while polyclonal IgGs (pIgGs) from non-immunized rabbits and animals immunized with BSA were catalytically inactive. It was shown that 74-85% of the total IgG DNase and RNase activities belongs to anti-idiotypic antibodies to RNase A (0.6-0.8% of total pIgGs), while 15-26% of the activities cannot interact with Sepharose-bearing antibodies against RNase A and may be antibodies to nucleic acids bound to RNase. Affinity chromatography on DNA-cellulose separated catalytic IgGs into several antibody subfractions demonstrating only DNase or RNase activity or hydrolyzing RNA faster than DNA. Our data suggest that a fraction of abzymes (or catalytically active antibodies) from AI patients hydrolyzing both DNA and RNA may be antibodies against RNase or its complexes with other proteins.

Immunization of rabbits with DNase I produces polyclonal antibodies with DNase and RNase activities

Immunization of rabbits with DNase I leads to the production of antiidiotypic Abs with DNase activity. It is not known at present whether antiidiotypic Abs against DNA-hydrolyzing enzymes can possess RNase activity. Here we show that immunization of healthy rabbits with bovine DNase I produces IgGs with intrinsic DNase and RNase activities. Electrophoretically and immunologically homogeneous polyclonal IgGs were obtained by sequential chromatography of the immune sera on Protein A-Sepharose and gel filtration. Affinity chromatography on DNA cellulose using elution of Abs with different concentrations of NaCl and an acidic buffer separated catalytic IgGs into four Ab subfractions, three of which demonstrated only DNase activity while one subfraction hydrolyzed RNA faster than DNA. The serum of patients with many different autoimmune (AI) diseases contains small fractions of antibodies (Abs) interacting with immobilized DNA, which possess both DNase and RNase activities. Our data suggest that a fraction of abzymes from AI patients hydrolyzing both DNA and RNA can contain a subfraction of Abs against DNase I.

Catalytic IgG from Patients with Hemophilia A Inactivate Therapeutic Factor VIII

Factor VIII (FVIII) inhibitors are anti-FVIII IgG that arise in up to 50% of the patients with hemophilia A, upon therapeutic administration of exogenous FVIII. Factor VIII inhibitors neutralize the activity of the administered FVIII by sterically hindering its interaction with molecules of the coagulation cascade, or by forming immune complexes with FVIII and accelerating its clearance from the circulation. We have shown previously that a subset of anti-factor VIII IgG hydrolyzes FVIII. FVIII-hydrolyzing IgG are detected in over 50% of inhibitor-positive patients with severe hemophilia A, and are not found in inhibitor-negative patients. Although human proficient catalytic Abs have been described in a number of inflammatory and autoimmune disorders, their pathological relevance remains elusive. We demonstrate here that the kinetics of FVIII degradation by FVIII-hydrolyzing IgG are compatible with a pathogenic role for IgG catalysts. We also report that FVIII-hydrolyzing IgG from each patient exhibit multiple cleavage sites on FVIII and that, while the specificity of cleavage varies from one patient to another, catalytic IgG preferentially hydrolyze peptide bonds containing basic amino acids.

High levels of catalytic antibodies correlate with favorable outcome in sepsis

Sepsis is the leading cause of death in intensive care units and results from a deleterious systemic host response to infection. Although initially perceived as potentially deleterious, catalytic antibodies have been proposed to participate in removal of metabolic wastes and protection against infection. Here we show that the presence in plasma of IgG endowed with serine protease-like hydrolytic activity strongly correlates with survival from sepsis. Variances of catalytic rates of IgG were greater in the case of patients with severe sepsis than healthy donors (P < 0.001), indicating that sepsis is associated with alterations in plasma levels of hydrolytic IgG. The catalytic rates of IgG from patients who survived were significantly greater than those of IgG from deceased patients (P < 0.05). The cumulative rate of survival was higher among patients exhibiting high rates of IgG-mediated hydrolysis as compared with patients with low hydrolytic rates (P < 0.05). An inverse correlation was also observed between the markers of severity of disseminated intravascular coagulation and rates of hydrolysis of patients' IgG. Furthermore, IgG from three surviving patients hydrolyzed factor VIII, one of which also hydrolyzed factor IX, suggesting that, in some patients, catalytic IgG may participate in the control of disseminated microvascular thrombosis. Our observations provide the first evidence that hydrolytic antibodies might play a role in recovery from a disease.

Hydrolysis of myelin basic protein by polyclonal catalytic IgGs from the sera of patients with multiple sclerosis

Various catalytic antibodies or abzymes have been detected recently in the sera of patients with several autoimmune pathologies, where their presence is most probably associated with autoimmunization. Recently we have shown that DNase, RNase, and polysaccharide-hydrolyzing activities are associated with IgGs from the sera of patients with multiple sclerosis (MS). Here we present evidence demonstrating that highly purified MS IgGs (but not Igs from the sera of healthy individuals) catalyze specifically hydrolysis of human myelin basic protein (hMBP). In contrast to many known proteases, IgGs do not hydrolyze many other different proteins. Specific inhibitors of acidic and thiol proteases have no remarkable effect on proteolytic activity of IgGs. However, specific inhibitor of serine (PMSF, AEBSF, and benzamidin) and metal-dependent (EDTA) proteases significantly inhibit activity of proteolytic abzymes. Interestingly, the ratio of serine-like and metal-dependent activities of MS IgGs varied very much from patient to patient. The findings speak in favor of the generation by the immune systems of individual MS patients of a variety of polyclonal anti-MBP IgGs with different catalytic properties.

Catalytic heterogeneity of polyclonal DNA-hydrolyzing antibodies from the sera of patients with multiple sclerosis

Various catalytic antibodies or abzymes have been detected recently in the sera of patients with several autoimmune pathologies, where their presence is most probably associated with autoimmunization. Recently we have shown that DNase activity is associated with IgGs from the sera of patients with multiple sclerosis (MS) but not with those from the sera of normal humans. Here we present evidence showing that MS IgG, its F(ab) fragments, and separated L-chains catalyze DNA hydrolysis. The properties of the DNase activity of these polyclonal IgGs distinguish them from other known human DNases. In addition, their specific activities with different oligonucleotide substrates and the range of optimal pHs, apparent K M values and substrate specificities varied widely for different patients. The findings speak in favor of the generation by the immune systems of individual patients of a variety of polyclonal catalytic IgG pools, from relatively small to extremely large ones.

Catalytic mechanism of an abzyme displaying a beta-lactamase-like activity.

A catalytic IgG (Ab2) displaying a beta-lactamase-like activity was previously obtained by using the antiidiotypic pathway: the particularity of this antibody is that it is a true antiidiotype of the beta-lactamase active site. We have previously demonstrated that this IgG has retained some of the structural information displayed by the beta-lactamase active site, evident from data that polyclonal anti-Ab2 antibodies (Ab3) recognize beta-lactamase. In this article, we investigated the catalytic mechanism of the abzyme compared to that of the enzyme. The experimental data allow us to hypothesize the catalytic residues required for catalysis.

Low level formation of potent catalytic IgG fragments mediated by disulfide bond instability

A highly purified preparation of a monoclonal antibody to vasoactive intestinal peptide (VIP) was analysed by gel filtration. Three peaks of VIP hydrolysing activity were observed, corresponding to the 150 kDa tetramer IgG, 80 kDa dimer of the heavy and light chains (H-L dimer) and 25 kDa L chain monomer. The hydrolytic activity of all three peaks was removed by adsorption on immobilized anti-mouse IgG. The specific activities (CPM hydrolysed/μg protein) of the H-L dimer and the L chain monomer were more than 30-fold greater than of intact IgG. The presence of small amounts of the antibody fragments in unreduced IgG preparations was confirmed by electrophoresis of overloaded radiolabeled and unlabeled IgG preparations. Increased levels of the fragments were evident after prolonged incubation of a dilute solution of 125I-IgG at 37°C. Iodoacetamide, a sulfahydryl alkylating reagent, suppressed the production of IgG fragments. Incubation of 125I-labeled L chain with unlabeled IgG resulted in incorporation of small amounts of the radioactivity into disulfide bonded 150 kDa IgG tetramer and 50 kDa L chain dimer fractions. These observations implicate disulfide reduction and exchange reactions as the mechanism underlying formation of the IgG fragments. Like the antibody fragments found in unreduced IgG, the L chain monomer and non-covalently associated H-L dimer isolated from reduced and alkylated IgG were capable of hydrolysing VIP. Hydrolysis of VIP by the recombinant L chain subunit was inhibited by excess IgG, suggesting that high affinity VIP binding by IgG limits its hydrolysis by the L chain. These observations sugest that small amounts of high activity antibody fragments may contribute to the catalytic characteristics of the anti-VIP IgG preparation.

Esterase activity of catalytic IgG and IgM antibodies for the hydrolysis of p-nitrophenyl acetate

The esterase activities of IgG and IgM monoclonal antibodies (Antibody KD2-1 to KD2-260) prepared with a hapten of p-nitrophenyl hydrogen 4-(hydroxycarbonyl) butylphosphonate were examined in the hydrolysis of p-nitrophenyl acetate in 2.4–4.0% (v/v) MeCNH 2O in TrisHCl buffer (pH 5.0–8.0) at 293–308 K. The most efficient Antibody KD2-260, which resulted in k cat = 2.53 (293 K)−7.91 min −1 (308 K), k cat/ k uncat = 2.09 × 10 3 (308 K)−3.12 × 10 3 (293 K), and K m = 4.9 × 10 −6 (293 K)−1.5 × 10 −5 mol·dm −3 (308 K) at pH 6.0, was found to be catalytically more active than the Schultz antibody (Antibody 48G-7-4A1) prepared with the same hapten. The K m/ K i ratio (80) obtained for the hydrolysis activity of Antibody KD2-260 at 303 K (pH 6.0) by the inhibition experiment, the value of which is corresponding to ( k cat/ k uncat)/29, demonstrated the efficient Antibody KD2-260 catalyzed hydrolysis via the stabilization of the transition state (TS) of the reaction. The analogy between TS and TS analogue (or the hapten) in their electronic structures and the activation parameters was also discussed in connection with the esterase activity of Antibody KD2-260.