Conformation of immunoglobulin M. I. Characterization of anti-epsilon-1-dimethylamino-5-naphthalenesulfonyl-L-lysine immunoglobulin M antibodies from horse, pig, and shark.
Abstract: IgM antibodies specific for the fluorophore epsiolon-1-dimethylamino-5-naphthalenesulfonyl-L-lysine(DNS-lysine) were elicited in the horse and nurse shark by immunization with a DNS-lysine streptococcal conjugate; the antibodies were purified by specific adsorption with an immunoadsorbent followed by gel filtration to select the IgM class (molecular weight 900 000). About 90% of the equine anti-DNS was IgM.DNS-Lysine, when bound in the combining sites of a population of these anti-DNS IgM antigodies from horse and nurse shark, as well as from pig, exhibited a marked fluorescence enhancement and shift of the emission spectrum to shorter wavelengths compared with emission in aqueous solution; these results indicate that the environments of the anti-DNS combining sites of this population were relatively hydrophovic. Approximately one-third of the ten possible combining sites in each of these anti-DNS IgM species bound DNS-lysine in this manner with an average intrinsic association constant (Ko) of greater than 10(6) M-1. Small differences were noted in binding behavior among the three species of antibodies. The enzymatic susceptibility of equine IgM was similar to that of human IgM. (Fab')2mu, Fab'mu, and Fabmu fragments were prepared following digestion with pepsin. These fragments could be clearly differentiated on the basis of molecular size. They bound DNS-lysine with the same affinity as intact IgM and the DNS-lysine-fragment complexes exhibited the same spectral properties as the parent IgM. It was concluded that the anti-dNs IgM antibodies from all three species, as well as the enzymatic fragments, were suitable for nanosecond depolarization studies which are reported in the accompanying paper Holowka, D.A., and Cathou, R.E. (1976), Biochemistry, the following papter in this issue.
Publication Date: 1976-07-27 PubMed ID: 986159DOI: 10.1021/bi00660a032Google Scholar: Lookup
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- Journal Article
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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This research investigates the properties and behaviors of immunoglobulin M (IgM), a type of antibody, when interacting with a specific substance in horses, pigs, and sharks.
Objective and Methodology
- The study’s main objective was to explore the behavior of immunoglobulin M (IgM) antibodies in different animal species (horse, pig, and shark) while interacting with a particular substance, epsiolon-1-dimethylamino-5-naphthalenesulfonyl-L-lysine (DNS-lysine).
- The selected animals were immunized with a DNS-lysine streptococcal conjugate. Post-immunization, the antibodies were purified through a process of specific adsorption using an immunoadsorbent and gel filtration.
- The researchers aimed to select the IgM class of antibodies, which are notably massive, with a molecular weight of around 900,000.
Findings and Interpretation
- After binding with DNS-lysine, the IgM antibodies showed a significant enhancement in fluorescence and a shift of the emission spectrum to shorter wavelengths. This suggests that the combining sites of these antibodies were relatively hydrophobic, earlier when compared with the emission in an aqueous solution.
- Within each of the IgM species, approximately one-third of the ten potential combining sites bound DNS-lysine, with an average intrinsic association constant (Ko) greater than 10(6) M-1.
- While there were small differences in binding behavior among the three species of antibodies, the enzymatic susceptibility of equine IgM was found to be similar to that of human IgM.
Preparation and Analysis of Enzymatic Digestion Fragments
- The researchers prepared certain fragments post-digestion of IgM with pepsin. The fragments identified were (Fab’)2mu, Fab’mu, and Fabmu.
- These fragments were successfully differentiated based on their molecular size and showed the same affinity for DNS-lysine as the intact IgM.
- The DNS-lysine-fragment complexes also exhibited similar spectral properties as the parent IgM.
Conclusion
- The outcome of the research concluded that anti-DNS IgM antibodies from all three species, along with the enzymatic fragments, were suitable to be used in nanosecond depolarization studies.
- A subsequent paper will elaborate on such studies based on the findings of this research.
Cite This Article
APA
Holowka DA, Cathou RE.
(1976).
Conformation of immunoglobulin M. I. Characterization of anti-epsilon-1-dimethylamino-5-naphthalenesulfonyl-L-lysine immunoglobulin M antibodies from horse, pig, and shark.
Biochemistry, 15(15), 3373-3379.
https://doi.org/10.1021/bi00660a032 Publication
Researcher Affiliations
MeSH Terms
- Animals
- Antibodies / isolation & purification
- Bacterial Vaccines
- Binding Sites, Antibody
- Dansyl Compounds / immunology
- Horses
- Immunoglobulin M / analysis
- Immunoglobulin M / isolation & purification
- Lysine / immunology
- Molecular Weight
- Peptide Fragments / analysis
- Sharks
- Spectrometry, Fluorescence
- Streptococcus / immunology
- Swine
Citations
This article has been cited 1 times.- Congy N, Mihaesco C. Binding properties of goat IgM anti-dinitrophenyl antibodies.. Immunology 1978 Aug;35(2):307-15.
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