Joseph J. Unruh

Secondary structural studies of bovine caseins: temperature dependence of β-casein structure as analyzed by circular dichroism and FTIR spectroscopy and correlation with micellization

Abstract To obtain a molecular basis for the similarities and dissimilarities in the functional, chemical, and biochemical properties between β-casein and the other caseins, three-dimensional models have been presented. Secondary structural prediction algorithms and molecular modeling techniques were used to predict β-casein structure. The secondary structure of bovine β-casein was re-examined using Fourier transform infrared and circular dichroism spectroscopies to test these predictions. Both methods predict a range of secondary structures for β-casein (28–32% turns, 32–34% extended) at 25°C. These elements were highly stable from 5 to 70°C as viewed by circular dichroism. More flexible conformational elements, tentatively identified as loops, helix and short segments of polyproline II, were influenced by temperature, increasing with elevated temperatures. Another view is that as temperature decreases, these elements are lost (cold denaturation). Several distinct transitions were observed by circular dichroism at 10, 33 and 41°C, and another transition, extrapolated to occur at 78°C. Calculations from analytical ultracentrifugation indicate that the 10, 33 and 41°C transitions occur primarily in the monomeric form of the protein. As β-casein polymers are formed, and increase in size, the transitions at higher temperature may reflect changes in the more flexible conformational elements as they adjust to changes in surface charge during polymer formation. The transition at 10°C may represent an actual general conformational change or cold denaturation. Over the range of temperatures studied, the sheet and turn areas remain relatively constant, perhaps forming a supporting hydrophobic core for the monomers within the micelle-like polymer. This interpretation is in accord with the known properties of β-casein, and those predicted from molecular modeling.

Analytical13C NMR: A rapid, nondestructive method for determining the cis,trans composition of catalytically treated unsaturated lipid mixtures

Abstract and SummaryHigh resolution natural abundance 13C Fourier transform nuclear magnetic resonance (NMR) has been found to be an effective tool for the rapid and direct determination of the cis/trans composition in partially hydrogenated and isomerized unsaturated lipids. With the eis and trans allylic carbon resonances as representative probes for double bond stereo-chemistry. Evaluation of the cis/trans composition of complex, positionally isomerized mixtures can be made without the necessity of carrying out detailed analyses of multishift olefinic carbon resonances. Migration of double bonds in monoenes and polyenes and formation of conjugated unsaturation in cata-lytically treated fats are discussed and assessed as possible sources of error in the evaluation of cis/trans isomer ratios. Carbon spin lattice relaxation times T1 were measured for both cis and trans allylic reson-ances in isomeric mixtures of varying composition to assure quantitative intensity relationships. 13C NMR compositional analysis of complex mixtures is demonstrated.

Quantitation of the global secondary structure of globular proteins by FTIR spectroscopy: Comparison with X-ray crystallographic structure

Fourier transform infrared spectroscopy (FTIR) is potentially a powerful tool for determining the global secondary structure of proteins in solution, providing the spectra are analyzed using a statistically and theoretically justified methodology. We have performed FTIR experiments on 14 globular proteins and two synthetic polypeptides whose X-ray crystal structures are known to exhibit varying types and amounts of secondary structures. Calculation of the component structural elements of the vibrational bands was accomplished using nonlinear regression analysis, by fitting both the amide I and amide II bands of the Fourier self-deconvoluted spectra, the second-derivative spectra, and the original spectra. The methodology was theoretically justified by comparing (via nonlinear regression analysis) the global secondary structure determined after deconvolving into component bands the vibrational amide I envelopes with the calculated structure determined by first principles from Ramachandran analysis of the X-ray crystallographic structure of 14 proteins from the Brookhaven protein data bank. Justification of the nonlinear regression analysis model with respect to experimental and instrumental considerations was achieved by the decomposition of all the bands of benzene and an aqueous solution of ammonium acetate into component bands while floating the Gaussian/Lorentzian character of the line shapes. The results for benzene yield all pure Lorentzian line shapes with no Gaussian character while the ammonium acetate spectra yielded all Gaussian line shapes with no Lorentzian character. In addition, all-protein spectra yielded pure Gaussian line shapes with no Lorentzian character. Finally, the model was statistically justified by recognizing random deviation patterns in the regression analysis from all fits and by the extra sum of squares F-test which uses the degrees of freedom and the root mean square values as a tool to determine the optimum number of component bands required for the nonlinear regression analysis. Results from this study demonstrate that the globular secondary structure calculated from the amide I envelope for these 14 proteins from FTIR is in excellent agreement with the values calculated from the X-ray crystallographic data using three-dimensional Ramachandran analysis, providing that the proper contribution from GLN and ASN side chains to the 1667 and 1650 cm(-1) component bands has been taken into account. The standard deviation of the regression analysis for the per cent helix, extended, turn and irregular conformations was found to be 3.49%, 2.07%, 3.59% and 3.20%, respectively.

Effects of carbohydrate-structure changes on induced shifts in differential isotope-shift 13C-N.M.R.

Abstract Deuterium-induced, 13C-isotope shifts are shown to vary considerably from the initially predicted values calculated for ordinary pyranose and furanose sugars, when minor structural changes are introduced into the carbohydrate ring. Both substitution of C-OH groups or reduction of C-OH to CH2 permitted the evaluation of γ effects of OD without the contribution of β-OD-induced shifting. The observed γ-shift values for these modified structures were twice as large as those previously noted. This difference is most probably due to favored salvation. Substitution of OH at C-6 led to the predicted loss of differential isotope-shift (d.i.s.) at C-6 because of its isolation from all β and γ OD groups. The 31P resonances of d -glucose 6-phosphate show downfield deuterium shifts. Based on d.i.s. values, new 13C-shift assignments are proposed for isomaltose and 2-amino-2-deoxy-α- d -glucose. A study of acidic carbohydrates has demonstrated that isotope shifts are somewhat larger for sp2-hybridized carbon atoms whose OH groups are acidic. Relaxation times for sp2 carbon atoms isolated from dipolar interaction with protons were very long in D2O relative to their relaxation time in the H2O environment.

Solid-phase extraction of sulfamethazine in milk with quantitation at low ppb levels using thin-layer chromatography.

Abstract A method for the solid-phase extraction and thin-layer chromatographic (TLC) quantitation of sulfamethazine (SMZ) residues in milk is presented. Sulfabromomethazine was added as an internal standard to homogenized milk samples which were then diluted and passed through C 18 solid-phase extraction columns. The C 18 columns were eluted with methanol, and interfering components in the methanol were removed by passing eluate over an acidic alumina column. The analyte was then concentrated on a small ion-exchange resin. SMZ was eluted and applied to a silica gel TLC plate. Fluorescence detection was induced with fluorescamine and quantitated with a scanning densitometer. Recoveries were 88.36–103.15% in the analysis range [0.51–15.34 ppb (μg/l)]. The average recovery over the analysis range was 96.07%, with a coefficient of variation of 12.52%.

The Importance of Effective Predevelopment Plate Washing Techniques in Thin-Layer Chromatography

Abstract Comparative studies were made of the two methods commonly used for predevelopment TLC plate cleaning. In separate studies TLC plates were washed by the two methods: ascending development and dip washing using several solvent and solvent combinations. Residues remaining on the surface of plates after washing were visualized by fluorescence techniques and scanned by densitometry. Evaluation of the two washing methods showed that plate dipping gave superior results in every case over ascending development. The most effective solvent for predevelopment TLC plate washing was found to be methanol.

Reproducibility of the nonlinear regression fit to the FTIR spectra of lysozyme.

Controversy exists concerning the influence of experimental artifacts on the number of component FTIR vibrational bands which may be resolved from the amide I and II envelopes of proteins in water. Whether these bands represent unique populations of vibrating protein groups in a particular global 2 degrees structure or whether the bands are due to instrumental and environmental fluctuations has been addressed, T.F. Kumosinski and J.J. Unruh, Talanta, 43 (1996) 199-219. The repeatability of the methodology and the apparent uniqueness of the nonlinear regression fits are addressed in this study. We obtained a series of the spectra of lysozyme, and carried-out nonlinear regression analysis of each spectrum. Coefficients of variation (COV) were calculated for the wavenumber and area values of assigned component peaks obtained. Low COVs obtained attest to the precision of the methodology and the apparent uniqueness of the nonlinear regression fits. This methodology for acquisition and analysis of protein FTIR spectra yields results with good precision.