Francis E. Stolzenbach

[53] Lactic dehydrogenases (crystalline)

Publisher Summary This chapter discusses the determination of crystalline lactic dehydrogenases. The methods developed in the laboratories for the purification of lactic dehydrogenases involve several basic steps: (1) fractionation by solubility, (2) separation by charge, and (3) separation by molecular size. The mainstay of all the procedures is fractionation by solubility, using ammonium sulfate. Gross precipitations for the purpose of concentrating and for purification are used in most of our procedures. Repeated ammonium sulfate fractionations using finer cuts as the purification progresses are utilized in most procedures. Separation by charge using cation and anion exchange celluloses is the other main purification step. During the course of electrophoretic investigation, it was noted that some proteins had their mobility changed drastically while others were unaffected by changes in pH. This helped greatly in determining the exchanger and pH to use in the procedure. Proteins which chromatograph as one symmetrical peak may be separated on a subsequent column simply by changing the pH conditions for equilibration and running of the column. Sephadex molecular sieves and sucrose density gradient centrifugations are used for separation by molecular size.

Immobilization of hydrogenase on glass beads.

Abstract Hydrogenase from Clostridium pasteurianum was immobilized on glass beads by four different methods. The sensitivity of the native and bound enzyme to oxygen was examined. Hydrogenase bound to succinyl glass proved to be the most stable to oxygen. All bound enzymes were active with ferredoxin as a substrate and evolved hydrogen in a chloroplast-ferredoxin-hydrogenase system driven by light.

Immobilized enzymes: The catalytic properties of lactate dehydrogenase covalently attached to glass beads

Abstract Chick LDH (H4 and M4) has been covalently attached to aryl and alkyl amine glass using sodium nitrite and glutaraldehyde respectively. These immobilized enzymes remain active for months at 0°C and exhibit Km values similar to those of the soluble enzyme; however, they have pH-rate profiles that are independent of pH and show decreased substrate inhibition. Disaggregation followed by reassociation indicate the enzymes are bound by all four subunits and the resulting activity restored to the native, aryl amine and glutaraldehyde bound enzyme are 33, 25 and 90% respectively. At a pH of 3.2 and 25°, the soluble and aryl amine glass LDHs are rapidly denatured while the glutaraldehyde bound enzyme shows no loss of activity for at least 35 days.

Mitochondrial malate dehydrogenase: Further studies on multiple electrophoretic forms

Abstract Previous studies on chicken heart mitochondrial malate dehydrogenase suggested that the multiple electrophoretic forms differed only in conformation. Further experiments have now shown that, while it is possible to effect marked changes in the electrophoretic mobility of the various forms as previously described by extended reversible denaturation in either acid media or concentrated guanidine hydrochloride, the products of these treatments differ from the native enzyme in thermal stability.

[64] Immobilization of lactic dehydrogenase☆

Publisher Summary This chapter discusses the method of immobilization of lactic dehydrogenase. The enzyme immobilization in laboratories can been done using porous-glass beads that have been derivatized to yield alkylamine, arylamine, and succinyl glass. Alkylamine glass can also be used with a bifunctional reagent to cross-link the enzyme to the glass. These procedures are carried out under conditions where, there was not a great excess of enzyme and yielded products in which, the enzyme was linked by all four subunits and most likely at several residues on each subunit. The physical and catalytic properties of these enzymes exhibit very interesting and different characteristics from the native soluble enzyme. Details of binding to Sepharose and porous-glass derivatives and the preparation of the derivatized glass are covered in chapter.