Carboxymethyl cellulose as a new heterobifunctional ligand carrier for affinity precipitation of proteins

Affinity precipitation is a technique that imparts selectivity to the widely used primary purification step of precipitation of proteins from crude extracts. Hetero-bifunctional affinity precipitation involves use of reversibly soluble/insoluble polymers that can be used as backbones to conjugate affinity ligands for specific separations. A variety of such polymers have been reported in literature. In this work we report development of carboxymethyl cellulose (CM cellulose) as a cheap, readily available and versatile reversibly soluble polymer system. Available CM cellulose as sodium salt could be quantitatively precipitated from its aqueous solution in presence of about 50 mM calcium and 7.2% w/v polyethylene glycol-4000, and could be resolubilised in the working buffer in absence of calcium, polyethylene glycol or both. Effectiveness of the CM cellulose-calcium-polyethylene glycol system was demonstrated by purifying lactate dehydrogenase from porcine muscle extractusing covalently conjugated Cibacron blue dye-ligand. By careful choice of conditions that suppressed non-specific interactions, the system was shown to be an effective affinity precipitation polymer system inspite of the polyelectrolytic nature of CM cellulose. Up to 23 fold purification of the enzyme from crude extarct was obtained in one single precipitation sequence. This revised version was published online in July 2006 with corrections to the Cover Date.     

Affinity isolation of a cold-adapted enzyme: lactate dehydrogenase from Bacillus psychrosaccharolyticus

A simple, economical and rapid affinity chromatography procedure with dyes as the ligand has been described for the one-step purification of a cold-adapted lactate dehydrogenase. Non-specific elution of Procion blue H-ERD-modified Sepharose yielded homogeneous preparations of lactate dehydrogenase both in column based procedures and in batch wise operations. Low operational temperatures resulted in the enhanced binding of the enzyme to the blue dye. The dissociation constants of the enzyme-dye complexes were 7.2±0.2 M and 11.2±0.2 M at 5 °C and 20°C respectively.This revised version was published online in October 2005 with corrections to the Cover Date.     

Affinity purification and subunit structure of soya bean lactate dehydrogenase

The lactate dehydrogenase (LDH) from soya bean has been purified to homogeneity by affinity chromatography. The enzyme was purified by sequential adsor     

The amino acid composition of soya lactate dehydrogenase

Lactate dehydrogenase from germinating soya plants was prepared in an electrophoretically homogeneous form by affinity chromatography on AMP-Sepharose.     

Detection of lactate dehydrogenase B4 in human hemolysate of patients deficient in lactate dehydrogenase B subunit activity using enzyme immunoassay

We developed a sensitive enzyme immunoassay system specific for human lactate dehydrogenase (LDH)- B₄ with antiacetylated LDH-B₄ Fab-horse-radish peroxidase conjugate. The enzyme immunoassay system was not interfered with by up to 0.3 mg/tube of hemoglobin. Thus, we measured LDH-B₄ concentrations in the hemolysate of seven heterozygous individuals deficient in LDH-B subunit activity and eight normal individuals. We could not find a significant difference between the LDH-B₄ concentrations in heterozygous and those in normal individuals. These results demonstrate that heterozygous individuals deficient in LDH-B subunit activity produce enzymatically inactive B subunits.This work was supported in part by grants in aid for Scientific Research from the Ministry of Education, Japan (59570998), and from the Clinical Pathology Research Foundation of Japan.     

Tandem application of cationic colloidal silica and Triton X-114 for plasma membrane protein isolation and purification: towards developing an MDCK protein database

Plasma membrane (PM) proteins are attractive therapeutic targets because of their accessibility to drugs. Although genes encoding PM proteins represent 20-30% of eukaryotic genomes, a detailed characterisation of their encoded proteins is underrepresented, due, to their low copy number and the inherent difficulties in their isolation and purification as a consequence of their high hydrophobicity. We describe here a strategy that combines two orthogonal methods to isolate and purify PM proteins from Madin Darby canine kidney (MDCK) cells. In this two-step method, we first used cationic colloidal silica (CCS) to isolate adherent (Ad) and non-adherent (nAd) PM fractions, and then subjected each fraction to Triton X-114 (TX-114) phase partitioning to further enrich for hydrophobic proteins. While CCS alone identified 255/757 (34%) membrane proteins, CCS/TX-114 in combination yielded 453/745 (61%). Strikingly, of those proteins unique to CCS/TX-114, 277/393 (70%) had membrane annotation. Further characterisation of the CCS/TX-114 data set using Uniprot and transmembrane hidden Markov model revealed that 306/745 (41%) contained one or more transmembrane domains (TMDs), including proteins with 25 and 17 TMDs. Of the remaining proteins in the data set, 69/439 (16%) are known to contain lipid modifications. Of all membrane proteins identified, 93 had PM origin, including proteins that mediate cell adhesion, modulate transmembrane ion transport, and cell-cell communication. These studies reveal that the application of CCS to first isolate Ad and nAd PM fractions, followed by their detergent-phase TX-114 partitioning, to be a powerful method to isolate low-abundance PM proteins, and a useful adjunct for in-depth cell surface proteome analyses.     

Expression of lactate dehydrogenase isozyme 5 (LDH-5) in cultured mouse blastocysts in the absence of implantation and outgrowth

Extensive extraction studies with Triton X-100 revealed only LDH-1 (B₄) but no trace of LDH-5 (A₄) in one-cell and two-cell mouse and rat embryos. The LDH isozyme pattern of preimplantation mouse embryos changes from the maternally inherited B subunit isozyme (LDH-1) to a pattern dominated by LDH-5 when mouse blastocysts are cultured under conditions that prevent hatching but allow trophoblast giant cell transformation. During differentiation of mouse blastocysts in vitro, implantation is therefore not essential for the appearance of the A subunit form of LDH (LDH-5) coded for by the embryonic genome. Mechanisms controlling the expression of LDH-5 in mouse blastocysts during in vivo development are discussed.This work was supported by grants of the Deutsche Forschungsgemeinschaft awarded to the Sonderforschungsbereich 29—Embryonale Entwicklung and Differenzierung.     

Fluorimetric quantification of intracellular lactate dehydrogenase during fermentation using flow injection analysis

A flow injection system for the on-line detection of the intracellular enzyme lactate dehydrogenase (LDH) during fermentation has been developed. The system is comprised of an on-line cell disintegration part, an immobilised dye based expanded bed column for the affinity capture of LDH and a fluorimetric detection unit. The system with a linearity of 0.1–5.4 U LDH ml^–1 was applied for the detection of intracellular accumulation of LDH during Lactococcus lactis subsp.lactis cultivation.     

Analysis of glycyrrhizic acid and liquiritin in liquorice root with microwave-assisted micellar extraction and pre-concentration

The feasibility of employing a non-ionic surfactant (Triton X-100) as an alternative and effective solvent for the microwave-assisted extraction of glycyrrhizic acid and liquiritin from liquorice root has been demonstrated. When compared with commonly used solvents, 5% Triton X-100 yielded higher extraction efficiency than aqueous solutions of ethanol or methanol. Under optimal conditions, i.e. 5% Triton X-100 (v/v) and microwave-assisted extraction for 3-5 min at 100 degrees C, the percentage extraction of active ingredients reached the highest value. The pre-concentration factor for the glycyrrhizic acid and liquiritin was about 13, and the cloud-point extraction recoveries for the two ingredients were 98.4 and 96.1%, respectively. The results showed that the coupling of microwave-assisted extraction and cloud-point extraction could be employed as a new and effective approach for the rapid extraction and pre-concentration of pharmacologically active ingredients from liquorice root without disturbing the subsequent chromatographic analysis.     

Membrane and membrane-associated proteins in Triton X-114 extracts of Mycobacterium bovis BCG identified using a combination of gel-based and gel-free fractionation strategies

Tuberculosis is an ancient disease that remains a significant global health problem. Because many membrane and membrane-associated proteins of this pathogen represent potential targets for drugs, diagnostic probes or vaccine components, we have analysed Mycobacterium bovis, bacillus Calmette-Guérin (BCG) substrain Moreau, using Triton X-114 for extraction of lipophilic proteins, followed by identification with LC coupled MS/MS. We identified 351 different proteins in total, and 103 (29%) were predicted as integral membrane proteins with at least one predicted transmembrane region and another 84 (23.9%) proteins had a positive grand average of hydropathicity (GRAVY) value, indicating increased probability for membrane association. Altogether 43 predicted lipoproteins (Lpps) were identified which is close to 50% of the total number of Lpps in the genome. Fifty-four proteins, including twenty-four predicted integral membrane proteins and seven predicted Lpps are described for the first time. The proportion of hydrophobic membrane and membrane-associated proteins shows that Triton X-114 is a highly efficient method for extraction of membrane proteins from bacteria, without the need for preisolation of membranes. ATP synthase, NAD(P) transhydrogenase, ubiquinone oxidoreductase and ubiquinol-cytochrome C reductase appear to represent major enzyme complexes in the membrane of Mycobacterium tuberculosis complex organisms.     

Concentration of mammalian genomic DNA using two‐phase aqueous micellar systems

The concentration of biomarkers, such as DNA, prior to a subsequent detection step may facilitate the early detection of cancer, which could significantly increase chances for survival. In this study, the partitioning behavior of mammalian genomic DNA fragments in a two‐phase aqueous micellar system was investigated using both experiment and theory. The micellar system was generated using the nonionic surfactant Triton X‐114 and phosphate‐buffered saline (PBS). Partition coefficients were measured under a variety of conditions and compared with our theoretical predictions. With this comparison, we demonstrated that the partitioning behavior of DNA fragments in this system is primarily driven by repulsive, steric, excluded‐volume interactions that operate between the micelles and the DNA fragments, but is limited by the entrainment of micelle‐poor, DNA‐rich domains in the macroscopic micelle‐rich phase. Furthermore, the volume ratio, that is, the volume of the top, micelle‐poor phase divided by that of the bottom, micelle‐rich phase, was manipulated to concentrate DNA fragments in the top phase. Specifically, by decreasing the volume ratio from 1 to 1/10, we demonstrated proof‐of‐principle that the concentration of DNA fragments in the top phase could be increased two‐ to nine‐fold in a predictive manner. Biotechnol. Bioeng. 2009;102: 1613–1623. © 2008 Wiley Periodicals, Inc.