P-Galactosidases

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

Lactase (P-galactosidase) is produced commercially from the lactose fermenting Klujveromjcesfragilis. The enzyme has a pH optimum of 6—7 and is used ia the hydrolysis of lactose ia milk or skim milk. 

Goebel and Avery J Exptl Medicine 50 521 7929 Snyder and Link J Am Chem Soc 75 1758.] chromogenic substrate for P-galactosidases [Buoncore et al. J Appl Biochem 2 390 1980]. 

Husum et al. found that the hydrolytic activities of P-galactosidase from E. coli and the protease subtilisin in a 50 % aqueous solution of the water-miscible ionic liquid [BMIM][Bp4] were comparable to those in 50 % aqueous solutions of ethanol or acetonitrile (Entry 9) [37]. 

As in the alkaline phosphatase example above, p-galactosidase, an enzyme with a molecular weight of approximately 360,000, has also been incorporated into a polyanhydride and released in a well-controlled fashion. As is shown in Fig. 14, the release of 3-galactosidase was quite linear over most of the time examined, and was complete, reaching 100% release in about 800 hr. This experiment utilized 5% loaded, compression-molded wafers of PCPP-SA 9 91, 1.4 cm in diameter and 0.5 mm thick, weighing 50 mg. 

Removal of Gal from the RG-hydrolase MHR oligomers was performed as described in Mutter et al. (1994). From the RG-lyase MHR oligomers the Gal was removed using 9.3 pg P-galactosidase per mg substrate. Derhamnosylation of the degalactosylated RG-hydrolase MHR oligomers was carried out as described in Mutter et al. (1994). 

The authors wish to thank Maijo Searle-van Leeuwen for purification of the P-galactosidase from Aspergillus niger and Novo Nordisk Ferment Ltd Dittingen (Switzerland) for assistance in the purification of the RG-hydrolase and Novo Nordisk A/S Bagsvaerd (Denmark) for the gift of the crude recombinant RG-lyase. 

Determination of molecular mass of pectic enzymes The molecular mass were determined by gel filtration in a Sepharose CL-6B column (1,8 x 88cm) equilibrated and eluted with Tris-HCl 50 mM, pH 7,5 buffer, plus 100 mM KCl. Fractions (3,3 ml) were collected at a flow rate of 10 ml/h. Molecular mass markers were tyroglobulin (660 kDa) apoferritin (440 kDa) P-amylase (200 kDa) alcohol dehydrogenase (150 kDa) bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa). Urea-SDS-PAGE (7%) was carried out according to Swank and Munkres [12]. Molecular mass markers were myosin (205 kDa) p-galactosidase (116 kDa) phosphorylase b (97 kDa) bovine serum albumin (66 kDa), ovalbumin (45 kDa) and carbonic anhydrase (29 kDa). 

Figure 3. (A) Determination of molecular mass of pectic enzymes by gel filtration in Sepharose 6B. Molecular mass markers - tyroglobulin, 2- apoferritin, 3- p-amylase, 4-alcohol dehydrogenase, 5- bovine serum albumin, 6- carbonic anhydrase. (B) SDS-PAGE of pectolytic activities. Molecular mass markers 1- myosin, 2- p-galactosidase, 3- phosphorylase b, 4- bovine serum albumin, 5- ovalbumin, 6- carbonic anhydrase.

Ferric ion was immobilized on a Chelating Sepharose Fast Flow column preparatory to the separation of seven enkephalin-related phosphopep-tides.17 Non-phosphorylated peptides flowed through the column, and the bound fraction contained the product. The capacity of the column was found to be 23 pmol/mL by frontal elution analysis. Cupric ion was immobilized on Chelating Superose for the isolation of bovine serum albumin.18 Cupric ion was immobilized on a Pharmacia HiTrap column for the separation of Protein C from prothrombin, a separation that was used to model the subsequent apparently successful separation of Factor IX from prothrombin Factor IX activity of the eluate was, however, not checked.19 Imidazole was used as the displacement agent to recover p-galactosidase from unclarified homogenates injected onto a nickel-loaded IMAC column.20 Pretreatment with nucleases and cleaning in place between injections were required procedures. A sixfold purification factor was observed. 

Clemmit, R.H. and Chase, H.A., Immobilized metal affinity chromatography of P-galactosidase from unclarified Escherichia coli homogenates using expanded bed adsorption, /. Chromatogr. A, 874, 27, 2000. 

Parameters Radiometric proximity assays (SPA, Flashplate) Fluorescence polarization (FP) Time- resolved fluorescence (HTRF) Amplified luminescence (ALPHAScreen) Enzyme (p-galactosidase) complementation Electrochemilumines cence... 

Farkade VD, Harrison STL, Pandit AB (2006) Improved cavitational cell disruption following pH pretreatment for the extraction of P-galactosidase from Kluveromyces lactis. Biochem Eng J 31 25-30... 

The following protocol illustrates the use of SIAB in preparing antibody-enzyme conjugates using P-galactosidase. 

Add P-galactosidase to the activated antibody solution at a ratio of 4 mg of enzyme per mg of antibody. 

If P-galactosidase is used to conjugate with an SMCC-activated (strept)avidin, then there is no need to thiolate the enzyme, since it contains sulfhydryls in its native state (Fujiwara et al., 1988 Sivakoff and Janes, 1988). For conjugations using HRP, alkaline phosphatase, or glucose oxidase, however, thiolation is necessary to add the requisite sulfhydryls. 

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