[63] Properties of detergents

This laboratory is investigating radiolabeled bacteriophages for specific detection of infection through gamma imaging. Previously a 99m Tc-labeled M13 phage demonstrated specific binding for its host Escherichia coli in vitro and in mice through imaging.Methods-This study was extended to phages P22, E79, VD-13, and phage 60. Each was radiolabeled with 99m Tc using the chelator MAG 3 , labeled phages were evaluated for binding to host and non host bacteria in vitro and in a mouse model. Results-In vitro each 99mTc-phage bound to its host at least 4-fold higher than to non host bacteria. For example, 99m Tc-E79 showed 10-to 20-fold greater binding to host Pseudomonas aeruginosa than non host Escherichia coli and Salmonella enterica, respectively. 99m Tc-phage 60 showed 20-fold greater binding to host Klebsiella pneumoniae over non hosts. Mice received host or non host bacteria in one thigh and 3 h later the 99m Tc-phages were administered iv. After a further 3 h the tissues were counted. Liver accumulation was highest for 99m Tc-E79, averaging 39% compared to an average of 13% for the other 99m Tc-phages. Animals infected with host bacteria showed infected thigh to normal thigh ratios of 14.2 for 99m Tc-E79, 2.9 for 99m Tc-P22, 3.5 for 99m Tc-VD-13 and 2.1 for 99m Tc-phage 60.Conclusions-Although specific host binding was observed in vitro for each of the these four 99m Tc-phages, only 99m Tc-E79 showed specificity for its host in an in vivo model.

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“…Since the detergent disrupts specific surface interactions (14), this reduction in binding is further evidence of specific binding (Fig. 2).…”

Section: Discussionmentioning

confidence: 86%

Investigation of four 99mTc-labeled bacteriophages for infection-specific imaging

Rusckowski

Gupta

Liu

et al. 2008

Nuclear Medicine and Biology

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“…The development of a rational approach to the solubilization of membrane proteins always requires a review of the literature to determine the detergent type and the conditions that are generally used for the solubilization of the protein of interest (1,(3)(4)(5)(6)(7)(8)10). All assays were carried at 37ºC as described in Methods.…”

Section: Discussionmentioning

confidence: 99%

“…Several excellent reviews can be found on detergents, their physical properties and their use in the solubilization of membrane proteins (1)(2)(3)(4). Due to three important factors, non-ionic detergents are frequently used for the solubilization of protein membranes: i) their efficiency in breaking the lipid-lipid and lipid-protein interactions, ii) their inefficiency in weakening protein-protein interactions, and iii) their property of being less denaturing than the ionic detergents (3)(4)(5)(6).…”

Section: Introductionmentioning

confidence: 99%

Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

Santos

Lamas

Ciancaglini

2002

Braz J Med Biol Res

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SDS, C 12 E 8 , CHAPS or CHAPSO or a combination of two of these detergents is generally used for the solubilization of Na,K-ATPase and other ATPases. Our method using only C 12 E 8 has the advantage of considerable reduction of the time for enzyme purification, with rapid solubilization and purification in a single chromatographic step. Na,KATPase-rich membrane fragments of rabbit kidney outer medulla were obtained without adding SDS. Optimum conditions for solubilization were obtained at 4ºC after rapid mixing of 1 mg of membrane Na,K-ATPase with 1 mg of C 12 E 8 /ml, yielding 98% recovery of the activity. The solubilized enzyme was purified by gel filtration on a Sepharose 6B column at 4ºC. Non-denaturing PAGE revealed a single protein band with phosphomonohydrolase activity. The molecular mass of the purified enzyme estimated by gel filtration chromatography was 320 kDa. The optimum apparent pH obtained for the purified enzyme was 7.5 for both PNPP and ATP. The dependence of ATPase activity on ATP concentration showed high (K 0.5 = 4.0 µM) and low (K 0.5 = 1.4 mM) affinity sites for ATP, with negative cooperativity. Ouabain (5 mM), oligomycin (1 µg/ml) and sodium vanadate (3 µM) inhibited the ATPase activity of C 12 E 8 -solubilized and purified Na,KATPase by 99, 81 and 98.5%, respectively. We have shown that Na,KATPase solubilized only with C 12 E 8 can be purified and retains its activity. The activity is consistent with the form of (aß) 2 association.

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“…Detergents are extremely important in studies of biological membranes due to their ability to solubilize membrane proteins and receptors [1][2][3][4][5][6]. They are soluble amphiphiles, and above a critical concentration (strictly speaking, a narrow concentration range), known as the critical micelle concentration (CMC), self associate to form thermodynamically stable, non-covalent aggregates called micelles [7][8][9].…”

Section: Introductionmentioning

confidence: 99%