Selective recovery of lactate dehydrogenase using affinity foam.
Selective isolation of lactate dehydrogenase (LDH) from porcine muscle extract was studied using foam generated from the vigorous stirring of a non-ionic surfactant, Triton X-114 derivatized with Cibacron blue. The cloud point of the surfactant-dye conjugate was higher than that of the native Triton X-114, and also the foam prepared from the affinity surfactant was more rigid taking a longer time to collapse. The equilibrium dissociation constant between pure LDH and surfactant-dye conjugate was 5.0 microM as compared to the value of 2.2 microM for the enzyme and free dye as measured by differential spectroscopy. The isolation procedure involved mixing of the porcine muscle extract with the affinity foam, separating and collapsing the foam, and warming the solution formed to 37 degrees C to yield the surfactant-dye phase and an aqueous phase containing the enzyme. The effect of surfactant concentration and protein load on enzyme recovery and purification was investigated. Under optimal conditions, LDH was quantitatively recovered with high purification factor in a very short time. Both recovery and purification were higher when foam prepared from an equivalent mixture of surfactant-dye conjugate and unmodified surfactant was used. The selectivity of interaction between LDH and detergent-dye conjugate was confirmed by lowered recovery when NADH was included during the binding step.
Department of Biotechnology, Center for Chemistry & Chemical Engineering, Lund University, P.O. Box 124, Sweden.
Sheryl
S
Fernandes
Rajni
R
Hatti-Kaul
Bo
B
Mattiasson
D000345:Affinity Labels / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000187:drug effects / Q000201:enzymology*; D009243:NAD / Q000493:pharmacokinetics; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000737:chemistry / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013501:Surface-Active Agents / Q000737:chemistry; D013552:Swine; D014227:Triazines / Q000737:chemistry / Q000378:metabolism*
D000345:Affinity Labels; D013501:Surface-Active Agents; D014227:Triazines; D009243:NAD; D011092:Polyethylene Glycols; C012842:Cibacron Blue F 3GA; D017830:Octoxynol; C010615:Nonidet P-40; D007770:L-Lactate Dehydrogenase