Improved purification of human lactate dehydrogenase isoenzyme-3 and studies with its fluorescein isothiocyanate and biotin conjugates.
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.
Department of Clinical Chemistry, Onze Lieve Vrouwe Gasthuis, Amsterdam, The Netherlands.
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Weijers
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de Bruijn
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Mulder
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Kruijswijk
D000345:Affinity Labels; D000596:Amino Acids / Q000032:analysis; D000906:Antibodies / Q000276:immunology; D001360:Avidin; D001710:Biotin; D002845:Chromatography / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D004912:Erythrocytes / Q000201:enzymology*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006801:Humans; D007070:Immunoglobulin A / Q000032:analysis / Q000276:immunology; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D013861:Thiocyanates
D000345:Affinity Labels; D000596:Amino Acids; D000906:Antibodies; D000939:Epitopes; D005452:Fluoresceins; D007070:Immunoglobulin A; D007527:Isoenzymes; D013861:Thiocyanates; D001360:Avidin; D001710:Biotin; D007770:L-Lactate Dehydrogenase; D016650:Fluorescein-5-isothiocyanate