Multidimensional analysis of denatured milk proteins by hydrophobic interaction chromatography coupled to a dynamic surface tension detector.

Multidimensional analysis of denatured milk proteins is reported using high-performance liquid chromatography (HPLC) combined with dynamic surface tension detection (DSTD). A hydrophobic interaction chromatography (HIC) column (a TSK-Gel Phenyl-5PW column, TosoBiosep), in the presence of 3.0 M guanidine hydrochloride (GdmHCl) as denaturing agent is employed as the mobile phase. Dynamic surface tension is measured through the differential pressure across the liquid-air interface of repeatedly growing and detaching drops. Continuous surface tension measurement throughout the entire drop growth (50 ms to 4 s) is achieved, for each eluting drop of 4 s length, providing insight into both the kinetic and thermodynamic behavior of molecular orientation processes at the liquid-air interface. An automated calibration procedure and data analysis method is applied with the DSTD system, which allows two unique solvents to be used, the HIC mobile phase for the sample and a second solvent (water for example) for the standard, permitting real-time dynamic surface tension data to be obtained. Three-dimensional data is obtained, with surface tension as a function of drop time first converted to surface pressure, which is plotted as a function of the chromatographic elution time axis. Experiments were initially performed using flow injection analysis (FIA) with the DSTD system for investigating commercial single standard milk proteins (alpha-lactalbumin, beta-lactoglobulin, alpha-, beta-, kappa-casein and a casein mixture) denatured by GdmHCl. These FIA-DSTD experiments allowed the separation and detection conditions to be optimized for the HIC-DSTD experiments. Thus, the HIC-DSTD system has been optimized and successfully applied to the selective analysis of surface-active casein fractions (alpha s1- and beta-casein) in a commercial casein mixture, raw milk samples (cow's, ewe's and goat's milk) and other diary products (yogurt, stracchino, mozzarella, parmesan cheese and chocolate cream). The different samples were readily distinguished based upon the selectivity provided by the HIC-DSTD method. The selectivity advantage of using DSTD relative to absorbance detection is also demonstrated.

Hydrophobic interaction chromatography coupled with atomic fluorescence spectrometric detection Effect of the denaturation on the determination of thiolic proteins.

Hydrophobic interaction chromatography coupled online with chemical vapour atomic fluorescence spectrometry (HIC-CVGAFS) has been optimized for the analysis of thiolic proteins in denaturing conditions. Proteins are pre-column simultaneously denatured and derivatized in phosphate buffer solution containing 8.0moldm(-3) urea and p-hydroxymercurybenzoate (PHMB) and the derivatized denatured proteins are separated on a silica HIC Eichrom Propyl column in the presence of 8.0M urea in the mobile phase. Post-column online reaction of derivatized denatured proteins with bromine, generated in situ by KBr/KBrO(3) in HCl medium, allowed the fast conversion of the uncomplexed PHMB and of the PHMB bound to proteins to inorganic mercury also in presence of urea. Hg(2+), present in solution as Hg(2+)-urea complex, is selectively detected by AFS in a Ar/H(2) miniaturized flame after sodium borohydride reduction to Hg. Under optimized conditions, online bromine treatment gives a 100+/-2% recovery of both free and protein-complexed PHMB. Denatured glyceraldehyde-3-phosphate dehydrogenase, aldolase, lactate dehydrogenase, trioso phosphate isomerase and beta-lactoglobulin have been examined. As the sensitivity and limit of detection of proteins in the HIC-CVGAFS apparatus depends on number of SH groups reacting with PHMB, the denaturation process, which increases the number of PHMB-reactive thiolic groups in proteins, improves the analytical performances of the described system in protein analysis. The detection limit for the denatured proteins examined was found in the range of 10(-10)-10(-12)moldm(-3), depending on the considered protein, with linear calibration curves spanning over four decades of concentration.

Characterization of denatured proteins by hydrophobic interaction chromatography: a preliminary study.

In this preliminary study hydrophobic interaction chromatography (HIC) is proposed as a good tool in order to detect conformational changes induced by chemical denaturants in two globular proteins, cytochrome C (Cyt C) and myoglobin (MYO). Alterations in protein structure were manifested chromatographically by reproducible changes in peak heights, retention time, and appearance of multiple peaks. The HIC behavior of the two model proteins denatured by guanidinium thyocyanate (GdmSCN) was investigated, keeping constant various concentrations of urea in the mobile phase in a TSK-Gel Phenyl-5PW column (TosoBiosep). Suitable elution conditions provide evidence of the simultaneous presence of two denatured forms in the case of MYO, and sequential different denatured states of Cyt C.

Separation and determination of denatured alpha(s1)-, alpha(s2)-, beta- and kappa-caseins by hydrophobic interaction chromatography in cows', ewes' and goats' milk, milk mixtures and cheeses.

Caseins alpha(s1)-, alpha(s2)-, beta- and kappa- from raw cows', ewes' and goats' milk were separated and determined by hydrophobic interaction chromatography (HIC) by using a Propyl column (Eichrom) in the presence of 8.0 M urea in the mobile phase. The method is based on fast and easy solubilization of real raw samples by 4.0 M guanidine thiocyanate followed by the HIC analysis, without any preliminary precipitation or separation of the casein fraction. Elution conditions have been optimized by analyzing commercial single bovine standard caseins and their mixture. In the optimized chromatographic conditions the four casein fractions were separated in less than 45 min. A linear relationship between the concentration of casein and peak area (UV absorbance detector at 280 nm) has been obtained over the concentration range of 0.5 to 40 microM. The detection limit for alpha-, beta- and kappa-caseins ranged between 0.35 and 0.70 microM. The precision of the method was evaluated, the coefficient of variation for alpha-, beta- and kappa-casein determination ranging between 3.0 and 6.0%. The method has been validated by the analysis of reference skim milk powder (BCR-063R) certificated for total nitrogen content. The method was applied to commercial casein mixture and to the qualitative and quantitative analysis of casein fractions in unprocessed, raw cows', goats' and ewes' milk (10 samples analyzed for each species), in one sample of unprocessed buffalos' milk and in commercial cheeses (mozzarella, robiola, ricotta and stracchino). Binary mixtures of milk (cow/goat and cow/ewe) were also analyzed and the ratio between casein peak areas (alpha(s1)/kappa, alpha(s2)/beta, beta/kappa and alpha(s2)/alpha(s1)) of the HIC chromatograms was proposed and discussed in order to evaluate a possible application of this method to detect milk adulteration.

New chromatographic method for separation and determination of denatured alphaS1-, alphaS2-, beta- and kappa-caseins by hydrophobic interaction chromatography.

Separation and determination of denatured alpha-, beta- and kappa-caseins by hydrophobic interaction chromatography (HIC) was improved by using a TSK-Gel Ether-5PW column (Tosoh Biosep). The method, already proposed and performed by a TSK-Gel Phenyl-5PW column (Tosoh Biosep), is based on fast and easy solubilization of commercial and real samples by 4.0 M guanidine thiocyanate and HIC analysis in the presence of 8.0 M urea in the mobile phase. Employment of the less hydrophobic ether phase had the main advantage of separating casein fractions in less than 22 min and, additionally, of separating a-casein in kappaS1- and alphaS2 -casein fractions. The method has been validated by the analysis of reference skim milk powder (BCR-063R) certified for total nitrogen content. A linear relationship between the concentration of casein and peak area (UV absorbance detector at 280 nm) has been obtained over the concentration range of 0.5-40 microM. The detection limit for alpha-, beta- and kappa-caseins ranged between 0.33 and 0.65 microM. The precision of the method was evaluated, the RSDs for alphaS1-, alphaS2-, beta- and kappa-casein determination ranging between 2.3 and 5.5% for standard solutions and between 4.4 and 6.2% for real sample solutions. The mean value of casein content found in eight aliquots of BCR-063R calculated with respect to the total protein content (estimated on the basis of certified total nitrogen content) was 78.3 +/- 6.1%. Results of linear fitting of standard additions data of alphaS1-, alphaS2-, beta- and kappa-caseins to BCR-063R were compared with linear fitting of alphaS1-, alphaS2-, beta- and kappa-casein calibration data. The method was applied to commercial caseins and to 30 real, raw samples. A statistical comparison was performed between results on quantitation of alpha-, beta- and kappa-caseins obtained by TSK-Gel Ether-5PW and TSK-Gel Phenyl-5PW HIC columns, showing more accurate results for chromatographic analysis performed by the ether column.

Antioxidant activity of flavonoids from Licania licaniaeflora.

In our screening program for antioxidants with DPPH radical scavenging activity we tested several flavonoids isolated from the leaves of Licania licaniaeflora (Chrysobalanaceae family) and identified by spectroscopic evidence, particularly with 1H and 13C NMR. All the isolated compounds exhibited DPPH radical scavenging activity: quercetin derivatives showed the strongest action, while the flavanone 8-hydroxy-naringenin and kaempferol 3-O-alpha-rhamnoside had the lowest.