Improved purification of human lactate dehydrogenase isoenzyme-3 and studies with its fluorescein isothiocyanate and biotin conjugates.

Abstract

Lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) isoenzyme-3 (LD-3) has been isolated in milligram quantities from human erythrocytes. Using an improved procedure--which involves complete hemolysis of the erythrocytes, diethylaminoethyl (DEAE)-Sephacel column chromatography, and 5'-AMP-Sepharose 4B affinity chromatography--we obtained 23,000-fold purified isoenzyme from the crude hemolysate (overall yield about 90%). The final product was homogeneous on polyacrylamide disc gel electrophoresis and had a specific activity of about 435 kU/g. Its amino acid composition is presented. With the eventual aim to make visible and isolate IgA kappa antibody-secreting B lymphocytes, we developed reproducible methods for preparing fluorescein isothiocyanate isomer-1-conjugated LD-3 with a fluorescein/LD-3 molar ratio between 1.3 and 3.3, and biotinylated LD-3 with a biotin/LD-3 molar ratio between 1.3 and 2.5. In evaluating the stability of these two conjugates, we determined that they still can react with IgA kappa to form the IgA kappa (LD-3)2 complex.

• Authors: R N Weijers, R de Bruijn, J Mulder, H Kruijswijk
• Journal: Clinical chemistry (Clin Chem) Vol. 36 Issue 1 Pg. 59-64 (Jan 1990) ISSN: 0009-9147 [Print] England
• PMID: 1688746 (Publication Type: Journal Article)

Chemical References

• Affinity Labels
• Amino Acids
• Antibodies
• Epitopes
• Fluoresceins
• Immunoglobulin A
• Isoenzymes
• Thiocyanates
• Avidin
• Biotin
• L-Lactate Dehydrogenase
• Fluorescein-5-isothiocyanate

Topics

• Affinity Labels
• Amino Acids (analysis)
• Antibodies (immunology)
• Avidin
• Biotin
• Chromatography (methods)
• Enzyme-Linked Immunosorbent Assay
• Epitopes (immunology)
• Erythrocytes (enzymology)
• Fluorescein-5-isothiocyanate
• Fluoresceins
• Humans
• Immunoglobulin A (analysis, immunology)
• Isoenzymes
• L-Lactate Dehydrogenase (immunology, isolation & purification, metabolism)
• Microscopy, Fluorescence
• Thiocyanates