Autofluorescence characterization of advanced glycation end products of hemoglobin

This article describes the analysis of autofluorescence of advanced glycation end products of hemoglobin (Hb-AGE). Formed as a result of slow, spontaneous and non-enzymatic glycation reactions, Hb-AGE possesses a characteristic autofluorescence at 308/345 nm (lambda(ex)/lambda(em)). Even in the presence of heme as a quenching molecule, the surface presence of the glycated adduct gave rise to autofluorescence with the quantum yield of 0.19. The specificity of monoclonal antibody developed against common AGE structure with Hb-AGE was demonstrated using reduction in fluorescence polarization value due to increased molecular volume while binding. The formation of fluorescent adduct in hemoglobin in the advanced stage of glycation and the non-fluorescent HbA(1c) will be of major use in distinguishing and to know the past status of diabetes mellitus. While autofluorescence correlated highly with HbA(1c) value under in vivo condition (r = 0.85), it was moderate in the clinical samples (r = 0.55). The results suggest a non-linear relation between glycemia and glycation, indicating the application of Hb-AGE as a measure of susceptibility to glycation rather than glycation itself.     

Fragility by elastic incoherent neutron scattering

The present work furnishes an operative definition for the fragility degree by using elastic incoherent neutron scattering (EINS). Such a definition is based on the relation between viscosity, a macroscopic quantity, and the atomic mean-square displacement, which refers to a nanoscopic property. This procedure has been used to analyze a set of glass-forming systems and it allows to obtain a linear dependence of the fragility parameter M, obtained by EINS, on the fragility parameter m, obtained by viscosity measurements.     

Analytical methods used to measure acrylamide concentrations in foods

The state-of-the-art of analysis for acrylamide in food is reviewed. The majority of analytical methods adopts a similar approach: addition of internal standard to the specimen, extraction with water, purification of extract using a solid-phase extraction cartridge, and then determination using either gas chromatography coupled to mass spectroscopy (GC/MS) after bromination, or direct measurement with liquid chromatography coupled to mass spectroscopy (LC/MS). The available methods generally show good agreement and are likely to be accurate. However, improvements in precision (within-laboratory) and repeatability (between-laboratory) are needed by particular data users.     

Separation of N2-ethyl-2'-deoxyguanosine-5'-monophosphate and four native deoxyribonucleoside monophosphates using capillary zone electrophoresis with polyethylene glycol as buffer additive

We investigated the separation of five deoxyribonucleoside monophosphates: 2'-deoxyguanosine-5'-monophosphate (dGMP), 2'-deoxyadenosine-5'-monophosphate (dAMP), 2'-deoxycytosine-5'-monophosphate (dCMP), 2'-deoxythymidine-5'-monophosphate (dTMP) and a dGMP adduct possessing N2-ethyl-guanine, which has been noted in relation to mutagenesis of alcohol, using capillary zone electrophoresis (CZE). The concentration of polyethylene glycol (PEG) as a modifier and the pH of the running solutions can efficiently control the observed separation. Interaction of PEG with analytes was quantitatively evaluated. PEG worked effectively as a hydrophobic selector in these separations. The values of pKa of the acidic-NH-groups in the base moieties of dGMP, dTMP, and the dGMP adduct are close to that of boric acid used as buffer of the running solutions. The control of their charge was facilitated, enabling improved separations. A more sufficient and fast separation was achieved by both optimization of pH of the running solutions and PEG concentration compared with that obtained by pH control alone. On-line concentration using a stacking method followed by the PEG-assisted CZE was briefly studied.     

Differential signal detection system for improved analysis of overlapping signals in liquid chromatography

We have developed a differential signal detection system for the analysis of liquid chromatographic signals using two or more detectors and a differential amplifier circuit. The proposed detection system is an improvement on conventional chromatography in which signals are detected by means of a single detector. The differential signal detection system eliminates difficulties of isolating minor components of the signal which are masked by the major component, as well as difficulties of separating two components of the signal having similar retention times.     

Application of the slotted quartz tube in flow-injection flame atomic-absorption spectrometry

The slotted quartz has been applied to flow-injection flame atomic-absorption spectrometry (FI-FAAS) showing several important advantages. The tube life was improved by a factor of 5-6 compared to conventional continuous aspiration. Flow impact systems were found not to be necessary in the applications so that larger enhancement factors may be achieved without sacrifice in precision. For 1.0 mg/l. copper, 1.0 mg/l. lead, 0.1 mg/l. cadmium and 1.0 mg/l. gold sensitivity enhancement factors of 3.1, 5.5, 5.3 and 4.0 were obtained with precisions of 1.3%, 1.1%, 1.6% and 1.7% RSD (n = 11) respectively. Application of the slotted quartz tube FI-FAAS method to the determination of heavy metals in urine has shown improved tolerance to interfering matrices. Recoveries obtained by spiking undiluted urine samples with 0.1 mg/l. copper and lead, and 0.01 mg/l. cadmium were in the range 100-102%.     

Remarkable proteolytic activity of imidazoles attached to cross-linked polystyrene

The insoluble resins synthesized by attaching imidazoles to poly(chloromethylstyrene-co-divinylbenzene) effectively hydrolyzed albumin with half-life as short as 20 min at pH 7 and 25 degrees C. Thus, peptide hydrolysis was accomplished with imidazole in an artificial system for the first time. The imidazole-based artificial proteinases manifested optimum activity at pH 7-8. The proteolytic activity of the imidazole-based artificial proteinases exceeded that of previously reported organic artificial proteinases including catalytic antibodies. High proteolytic activity was observed when imidazole was attached to the resin through the C-2 atom instead of the N atom. The catalytic activity was greatly reduced when the content of imidazole was lowered. This indicates catalytic cooperation of at least two proximal imidazole moieties attached to the resin. Possible mechanisms for the effective protein hydrolysis by the proximal imidazoles are presented.     

Stationary phase evaluations of quantum rate constants

We compute the quantum rate constant based on two extended stationary phase approximations to the imaginary-time formulation of the quantum rate theory. The optimized stationary phase approximation to the imaginary-time flux-flux correlation function employs the optimized quadratic reference system to overcome the inaccuracy of the quadratic expansion in the standard stationary phase approximation, and yields favorable agreements with instanton results for both adiabatic and nonadiabatic processes in dissipative and nondissipative systems. The integrated stationary phase approximation to the two-dimensional barrier free energy is particularly useful for adiabatic processes and demonstrates consistent results with the imaginary-time flux-flux correlation function approach. Our stationary phase methods do not require calculation of tunneling paths or stability matrices, and work equally well in the high-temperature and the low-temperature regimes. The numerical results suggest their general applicability for calibration of imaginary-time methods and for the calculation of quantum rate constants in systems with a large number of degrees of freedom.     

Chiral allyl cations are captured by furan with 100% stereoselectivity: synthesis of enantiopure 2-alkoxy-8-oxabicyclo

A low-temperature (-95 degrees C) protocol for intermolecular cycloadditions of furan to chiral silyloxyallyl cations in dichloromethane is described. Key precursors are open-chain, mixed a-ketoacetals, which are chiral. The resulting [4+3] cycloadducts are densely functionalized and are isolated as single enantiomers in high chemical yield. The yield of the cycloadducts increases with increasing dilution. Three and four stereogenic centres are created in one single step.     

Development of a bioreactor based on trypsin immobilized on monolithic support for the on-line digestion and identification of proteins

The preparation and characterization of a new trypsin-based bioreactor is here described for on-line protein digestion and peptide analysis. Trypsin was immobilized on an epoxy-modified silica monolithic support with a single reaction step and the amount of immobilized enzyme was found to be 66.07 mg (+/-11.75 S.D.)/column (n = 6). The bioreactor was coupled through a switching valve to an analytical column for the on-line digestion, peptide separation and identification of test proteins by ESI-MS-MS. The influence of various parameters (flow rate, temperature, buffer pH and molarity, etc.) on enzymatic activity was investigated by an experimental design and the mostly significant factor was found to be the flow rate. The efficacy of the reported on-line bioreactor for tryptic mapping is reported for somatostatin and myoglobin, selected as model compounds. Tryptic peptide maps obtained by on-line digestion of myoglobin were compared to those obtained by traditional off-line digestion. Sequence coverage obtained with the on-line protocol (21 peptides, 75.16% coverage of myoglobin sequence) was found to be comparable to the one obtained with the off-line protocol (18 peptides, 76.47% coverage). Sensitivity for myoglobin digestion and identification was 0.1 mg/ml. The reproducibily of the peptide maps in terms of retention time was from 1.53 to 4.31%, R.S.D.