1.Technical note: Analysis of total lipid and triacylglycerol content in small liver biopsy samples in cattle.
Starke A1, Haudum A, Busche R, Beyerbach M, Dänicke S, Rehage J. J Anim Sci. 2010 Aug;88(8):2741-50. doi: 10.2527/jas.2009-2599. Epub 2010 Mar 26.
A procedure is described for analyzing total lipid (TL) and triacylglycerol (TAG) in 2 sequential steps using small amounts (<100 mg) of bovine liver tissue. The TL was measured gravimetrically and TAG was measured enzymatically in the TL extract, using an automated analyzer. For gravimetric TL determination in milligrams per gram of liver fresh weight (FW), TL was extracted from homogenized tissue samples with hexane:isopropanol (at 20 degrees C, 24 h, constant agitation). The routine method was modified by adding a second hexane extraction step to optimize lipid extraction. The dry lipid extract was dissolved in hexane and aliquoted according to TL content for TAG analysis. An extra incubation period of 16 h was included for complete hydrolysis of TAG, using microbial lipase and nonaethylene glycol monododecyl ether detergent, before TAG was measured enzymatically using commercial test kits. Triolein was used as an internal standard.
2.Comparison of the different types of surfactants for the effect on activity and structure of soybean peroxidase.
Zhang W1, Dai X, Zhao Y, Lu X, Gao P. Langmuir. 2009 Feb 17;25(4):2363-8. doi: 10.1021/la803240x.
In the pH 2.6 and 5.2 systems, soybean peroxidase (SBP) (isoelectric point, pI 3.9) has positive and negative charge, respectively. In order to acquire detailed knowledge on the role played by electrostatics in the denaturation of proteins, a comparison of anionic surfactant sodium dodecyl sulfate (SDS), nonionic surfactant nonaethylene glycol monododecyl ether [C12H25O(CH2CH2O)9H] (AEO9), and cationic surfactant cetyltrimethylammonium bromide (CTAB) for the influences on the activity and structure of soybean peroxidase (SBP) was carried out by measuring the activity, far-UV circular dichrosm, fluorescence, and electronic absorption spectra of SBP in the pH 2.6 and 5.2 systems at 30 degrees C. In the pH 2.6 systems, the interaction of SDS with SBP results in an increase in the fluorescence intensity with a red shift of the emission maximum of the tryptophan fluorescence and a blue shift of the Soret band. In the meantime, the alpha-helix of SBP is unfolded and the activity of SBP is lost irreversibly.
3.Determination of critical micelle concentrations and aggregation numbers by fluorescence correlation spectroscopy: aggregation of a lipopolysaccharide.
Yu L1, Tan M, Ho B, Ding JL, Wohland T. Anal Chim Acta. 2006 Jan 18;556(1):216-25. Epub 2005 Oct 10.
Fluorescence correlation spectroscopy (FCS) is often used to determine the mass or radius of a particle by using the dependence of the diffusion coefficient on the mass and shape. In this article we discuss how the particle size of aggregates can be measured by using the concentration dependence of the amplitude of the autocorrelation function (ACF) instead of the temporal decay. We titrate a solution of aggregates or micelles with a fluorescent label that possesses a high affinity for these structures and measure the changes in the amplitude of the ACF. We develop the theory describing the change of the ACF amplitude with increasing concentrations of labels and use it to fit experimental data. It is shown how this method can determine the aggregation number and critical micelle concentration of a standard detergent nonaethylene glycol monododecyl ether (C12E9) and a lipopolysaccharide (LPS: Escherichia coli 0111:B4).
4.Dependence of the product chain-length on detergents for long-chain E-polyprenyl diphosphate synthases.
Pan JJ1, Ramamoorthy G, Poulter CD. Biochemistry. 2013 Jul 23;52(29):5002-8. doi: 10.1021/bi400681d. Epub 2013 Jul 11.
Long-chain E-polyprenyl diphosphate synthases (E-PDS) catalyze repetitive addition of isopentenyl diphosphate (IPP) to the growing prenyl chain of an allylic diphosphate. The polyprenyl diphosphate products are required for the biosynthesis of ubiquinones and menaquinones required for electron transport during oxidative phosphorylation to generate ATP. In vitro, the long-chain PDSs require addition of phospholipids or detergents to the assay buffer to enhance product release and maintain efficient turnover. During preliminary assays of product chain-length with anionic, zwitterionic, and nonionic detergents, we discovered considerable variability. Examination of a series of nonionic PEG detergents with several long-chain E-PDSs from different organisms revealed that in vitro incubations with nonaethylene glycol monododecyl ether or Triton X-100 typically gave chain-lengths that corresponded to those of the isoprenoid moieties in respiratory quinones synthesized in vivo.
