The use of circular dichroism in the investigation of protein structure and function

Circular Dichroism (CD) relies on the differential absorption of left and right circularly polarised radiation by chromophores which either possess intrinsic chirality or are placed in chiral environments. Proteins possess a number of chromophores which can give rise to CD signals. In the far UV region (240-180 nm), which corresponds to peptide bond absorption, the CD spectrum can be analysed to give the content of regular secondary structural features such as alpha-helix and beta-sheet. The CD spectrum in the near UV region (320-260 nm) reflects the environments of the aromatic amino acid side chains and thus gives information about the tertiary structure of the protein. Other non-protein chromophores such as flavin and haem moieties can give rise to CD signals which depend on the precise environment of the chromophore concerned. Because of its relatively modest resource demands, CD has been used extensively to give useful information about protein structure, the extent and rate of structural changes and ligand binding. In the protein design field, CD is used to assess the structure and stability of the designed protein fragments. Studies of protein folding make extensive use of CD to examine the folding pathway; the technique has been especially important in characterising molten globule intermediates which may be involved in the folding process. CD is an extremely useful technique for assessing the structural integrity of membrane proteins during extraction and characterisation procedures. The interactions between chromophores can give rise to characteristic CD signals. This is well illustrated by the case of the light harvesting complex from photosynthetic bacteria, where the CD spectra can be analysed to indicate the extent of orbital overlap between the rings of bacteriochlorophyll molecules. It is therefore evident that CD is a versatile technique in structural biology, with an increasingly wide range of applications.     

Quantitation of estrogens in ground water and swine lagoon samples using solid-phase extraction, pentafluorobenzyl/trimethylsilyl derivatizations and gas chromatography-negative ion chemical ionization tandem mass spectrometry

A method was developed for the confirmed identification and quantitation of 17beta-estradiol, estrone, 17alpha-ethynylestradiol and 16alpha-hydroxy-17beta-estradiol (estriol) in ground water and swine lagoon samples. Centrifuged and filtered samples were extracted using solid-phase extraction (SPE), and extracts were derivatized using pentafluorobenzy] bromide (PFBBR) and N-trimethylsilylimidazole (TMSI). Analysis was done using negative ion chemical ionization (NICI) gas chromatography-mass spectrometry-mass spectrometry (GC-MS-MS). Deuterated analogs of each of the estrogens were used as isotope dilution standards (IDS) and were added to the samples before extraction. A limit of quantitation of 1 ng/l in ground water was obtained using 500 ml of ground water sample, 1.0 ml of extract volume and the lowest calibration standard of 0.5 pg/microl. For a 25 ml swine lagoon sample, the limit of quantitation was 40 ng/l. The average recovery of the four estrogens spiked into 500 ml of distilled water and ground water samples (n = 16) at 2 ng/l was 103% (S.D. 14%). For 25 ml of swine lagoon samples spiked at 500, 1000 and 10,000 ng/l, the average recovery for the four estrogens was 103% (S.D. 15%). The method detection limits (MDLs) of the four estrogens spiked at 2 ng/l in a 500 ml of ground water sample ranged from 0.2 to 0.6 ng/l. In swine lagoon samples from three different types of swine operations, estrone was found at levels up to 25,000 ng/l, followed by estriol and estradiol up to levels at 10,000 and 3000 ng/l, respectively. It was found that pretreatment of swine lagoon samples with formaldehyde was necessary to prevent conversion of estradiol to estrone.     

Dielectric spectroscopy at microwave frequencies

A method is described which is capable of measuring the dielectric properties of liquids as a continuous function of frequency over the range 10 MHz to 18 GHz; an evaluation is made of its performance over the frequency range up to 4.2 GHz. The method described has substantial advantages in ease of operation and evaluation of results over the conventional “spot” frequency and time-domain spectrometer techniques.     

Molecular dynamics of confined glucose solutions

Silica gels containing solutions of glucose in heavy water at different concentrations have been prepared by a sol-gel method. Dynamical studies with quasielastic neutron scattering, compared with previous results on bulk solutions, show that the dynamics of the glucose molecules are not appreciably affected by the confinement, even though the gels behave macroscopically as solid materials. Small-angle neutron-scattering spectra on the same systems, fitted with a fractal model, yield a correlation length that decreases from 20 to 2.5 nm with increasing glucose concentration, suggesting a clustering of glucose molecules in concentrated solutions that is consistent with the dynamical measurements. These two sets of results imply that 20 nm is an upper limit for the scale at which the dynamics of glucose molecules in solution are affected by confinement.     

Signal and data processing for atomic absorption spectrophotometry

The state-of-the-art of signal and data processing techniques for atomic absorption spectrophotometry is described and discussed. Aspects of optical and atomizer design of greatest importance for providing the best signal to noise S/N ratio and minimum curvature are summarized. In background corrected systems, amplifier gain and time constants must be carefully matched, especially for transient signals. A method is given for calculating the sampling time of peak search systems. Methods of signal averaging are described and the importance of precision calculations is stressed. The correct sequence of readings for calibration is discussed. The causes of curvature are shown for simple and complex curves and methods of correction are compared. Other desirable functions are calculation of sensitivity and detection limit, error warnings and external data output facilities.