Classification of intact fresh plums according to sweetness using time-domain nuclear magnetic resonance and chemometrics

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Determination of quality parameters for mustard sauces in sealed packets using time-domain nuclear magnetic resonance spectroscopy and chemometrics

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Measuring the solubility product constant of paramagnetic cations using time-domain nuclear magnetic resonance relaxometry

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

High-throughput non-destructive nuclear magnetic resonance method to measure intramuscular fat content in beef

High intake of saturated fat from meats has been associated with cardiovascular disease, cancer, diabetes, and others diseases. In this paper, we are introducing a simple, high-throughput, and non-destructive low-resolution nuclear magnetic resonance method that has the potential to analyze the intramuscular fat content (IMF) in more than 1,000 beef portions per hour. The results can be used in nutritional fact labels, replacing the currently used average value. The method is based on longitudinal (T(1)) and transverse (T(2)) relaxation time information obtained by a continuous wave-free precession (CWFP) sequence. CWFP yields a higher correlation coefficient (r=0.9) than the conventional Carr-Purcell-Meiboom- Gill (CPMG) method (r=-0.25) for IMF in beef and is just as fast and a simpler pulse sequence than CPMG. The method can also be applied to other meat products.

Qualitative analysis by online nuclear magnetic resonance using Carr-Purcell-Meiboom-Gill sequence with low refocusing flip angles

The Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence has been used in many applications of magnetic resonance imaging (MRI) and low-resolution NMR (LRNMR) spectroscopy. Recently. CPMG was used in online LRNMR measurements that use long RF pulse trains, causing an increase in probe temperature and, therefore, tuning and matching maladjustments. To minimize this problem, the use of a low-power CPMG sequence based on low refocusing pulse flip angles (LRFA) was studied experimentally and theoretically. This approach has been used in several MRI protocols to reduce incident RF power and meet the specific absorption rate. The results for CPMG with LRFA of 3 pi/4 (CPMG(135)), pi/2 (CPMG(90)) and pi/4 (CPMG(45)) were compared with conventional CPMG with refocusing pi pulses. For a homogeneous field, with linewidth equal to Delta nu = 15 Hz, the refocusing flip angles can be as low as pi/4 to obtain the transverse relaxation time (T(2)) value with errors below 5%. For a less homogeneous magnetic field. Delta nu = 100 Hz, the choice of the LRFA has to take into account the reduction in the intensity of the CPMG signal and the increase in the time constant of the CPMG decay that also becomes dependent on longitudinal relaxation time (T(1)). We have compared the T(2) values measured by conventional CPMG and CPMG(90) for 30 oilseed species, and a good correlation coefficient, r = 0.98, was obtained. Therefore, for oilseeds, the T(2) measurements performed with pi/2 refocusing pulses (CPMG(90)), with the same pulse width of conventional CPMG, use only 25% of the RF power. This reduces the heating problem in the probe and reduces the power deposition in the samples. (C) 2011 Elsevier B.V. All rights reserved.

HR Nuclear Magnetic Resonance Spectroscopy for Authentication of Olive Oil Quality

The present work is a preliminary study to settle the optimum experimental conditions and data processing for accomplishing the strategies established by the Action Plan for the EU olive oil sector. The objectives of the work were: a) to monitor the evolution of extra virgin olive oil exposed to indirect solar light in transparent glass bottles during four months; b) to identify spectral differences between edible and lampant virgin olive oil by applying high resolution Nuclear Magnetic Resonance (HR-NMR) Spectroscopy. Pr esent study could contribute to determine the date of minimum storage, their optimum conditions, and to properly characterize olive oil.

The use of simultaneous H-1 & P-31 magic angle spinning nuclear magnetic resonance measurements to characterize energy metabolism during the conversion of muscle to meat

In order to investigate the potential of magic angle spinning nuclear magnetic resonance (MAS NMR) in the elucidation of post-mortem metabolism in muscle biopsies, simultaneous H-1 and (31)p MAS NMR measurements were made continuously on postmortem (20 min to 24 h) muscle longissimus samples from rabbits. The animals had either been or not been given adrenaline (0.5 mg kg(-1) 4 h pre-slaughter) to deplete stores of muscle glycogen. The intracellular pH was calculated from H-1 spectra, and the post-mortem rate of formation of lactate was followed and quantified. Comparison of measurements made on muscle samples from rabbits treated with adrenaline with measurements made on muscle samples from untreated' rabbits revealed significant effects of adrenaline treatment on both pH (pH24 h = 6.42 vs. pH24 It = 5.60) and formation of lactate (16 mmol g(-1) vs. 65 mmol g(-1)). The P-31 NMR spectra were used to follow the rate of degradation of ATP and phosphocreatine. The present study clearly shows that MAS NMR has potential for the study of post-mortem energy metabolism.

Salt diffusion and distribution in meat studied by Na-23 nuclear magnetic resonance imaging and relaxometry

This study introduces the use of combined Na-23 magnetic resonance imaging (MRI) and Na-23 NMR relaxometry for the study of meat curing. The diffusion of sodium ions into the meat was measured using Na-23 MRI on a 1 kg meat sample brined in 10% w/w NaCl for 3-100 h. Calculations revealed a diffusion coefficient of 1 x 10(-5) cm(2)/s after 3 h of curing and subsequently decreasing to 8 x 10(-6) cm(2)/s at longer curing times, suggesting that changes occur in the microscopic structure of the meat during curing. The microscopic mobility and distribution of sodium was measured using Na-23 relaxometry. Two sodium populations were observed, and with increasing length of curing time the relaxation times of these changed, reflecting a salt-induced swelling and increase in myofibrillar pore sizes. Accordingly, the present study demonstrated that pore size and thereby salt-induced swelling in meat can be assessed using Na-23 relaxometry.

Proton nuclear magnetic resonance investigation of the native and modified active site structure of heme proteins

Hemoproteins are a very important class of enzymes in nature sharing the essentially same prosthetic group, heme, and are good models for exploring the relationship between protein structure and function. Three important hemoproteins, chloroperoxidase (CPO), horseradish peroxidase (HRP), and cytochrome P450cam (P450cam), have been extensively studied as archetypes for the relationship between structure and function. In this study, a series of 1D and 2D NMR experiments were successfully conducted to contribute to the structural studies of these hemoproteins. ^ During the epoxidation of allylbenzene, CPO is converted to an inactive green species with the prosthetic heme modified by addition of the alkene plus an oxygen atom forming a five-membered chelate ring. Complete assignment of the NMR resonances of the modified porphyrin extracted and demetallated from green CPO unambiguously established the structure of this porphyrin as an NIII-alkylated product. A novel substrate binding motif of CPO was proposed from this concluded regiospecific N-alkylation structure. ^ Soybean peroxidase (SBP) is considered as a more stable, more abundant and less expensive substitute of HRP for industrial applications. A NMR study of SBP using 1D and 2D NOE methods successfully established the active site structure of SBP and consequently fills in the blank of the SBP NMR study. All of the hyperfine shifts of the SBP-CN- complex are unambiguously assigned together with most of the prosthetic heme and all proximal His170 resonances identified. The active site structure of SBP revealed by this NMR study is in complete agreement with the recombinant SBP crystal structure and is highly similar to that of the HRP with minor differences. ^ The NMR study of paramagnetic P450cam had been greatly restricted for a long time. A combination of 2D NMR methods was used in this study for P450cam-CN - complexes with and without camphor bound. The results lead to the first unequivocal assignments of all heme hyperfine-shifted signals, together with certain correlated diamagnetic resonances. The observed alternation of the assigned novel proximal cysteine β-CH2 resonances induced by camphor binding indicated a conformational change near the proximal side.^

Nuclear magnetic resonance spectroscopic and computational investigations of chloroperoxidase catalyzed regio- and enantio-selective transformations

Chloroperoxidase (CPO) is the most versatile heme-containing enzyme that catalyzes a broad spectrum of reactions. The remarkable feature of this enzyme is the high regio- and enantio-selectivity exhibited in CPO-catalyzed oxidation reactions. The aim of this dissertation is to elucidate the structural basis for regio- and enantio-selective transformations and investigate the application of CPO in biodegradation of synthetic dyes. ^ To unravel the mechanism of CPO-catalyzed regioselective oxidation of indole, the dissertation explored the structure of CPO-indole complex using paramagnetic relaxation and molecular modeling. The distances between the protons of indole and the heme iron revealed that the pyrrole ring of indole is oriented toward the heme with its 2-H pointing directly at the heme iron. This provides the first experimental and theoretical explanation for the "unexpected" regioselectivity of CPO-catalyzed indole oxidation. Furthermore, the residues including Leu 70, Phe 103, Ile 179, Val 182, Glu 183, and Phe 186 were found essential to the substrate binding to CPO. These results will serve as a lighthouse in guiding the design of CPO mutants with tailor-made activities for biotechnological applications. ^ To understand the origin of the enantioselectivity of CPO-catalyzed oxidation reactions, the interactions of CPO with substrates such as 2-(methylthio)thiophene were investigated by nuclear magnetic resonance spectroscopy (NMR) and computational techniques. In particular, the enantioselectivity is partly explained by the binding orientation of substrates. In third facet of this dissertation, a green and efficient system for degradation of synthetic dyes was developed. Several commercial dyes such as orange G were tested in the CPO-H2O 2-Cl- system, where degradation of these dyes was found very efficient. The presence of halide ions and acidic pH were found necessary to the decomposition of dyes. Significantly, the results revealed that this degradation of azo dyes involves a ferric hypochlorite intermediate of CPO (Fe-OCl), compound X.^

Determination of Biomolecular Interdomain Motions using Nuclear Magnetic Resonance

Biological macromolecules can rearrange interdomain orientations when binding to various partners. Interdomain dynamics serve as a molecular mechanism to guide the transitions between orientations. However, our understanding of interdomain dynamics is limited because a useful description of interdomain motions requires an estimate of the probabilities of interdomain conformations, increasing complexity of the problem.

Staphylococcal protein A (SpA) has five tandem protein-binding domains and four interdomain linkers. The domains enable Staphylococcus aureus to evade the host immune system by binding to multiple host proteins including antibodies. Here, I present a study of the interdomain motions of two adjacent domains in SpA. NMR spin relaxation experiments identified a 6-residue flexible interdomain linker and interdomain motions. To quantify the anisotropy of the distribution of interdomain orientations, we measured residual dipolar couplings (RDCs) from the two domains with multiple alignments. The N-terminal domain was directly aligned by a lanthanide ion and not influenced by interdomain motions, so it acted as a reference frame to achieve motional decoupling. We also applied {\it de novo} methods to extract spatial dynamic information from RDCs and represent interdomain motions as a continuous distribution on the 3D rotational space. Significant anisotropy was observed in the distribution, indicating the motion populates some interdomain orientations more than others. Statistical thermodynamic analysis of the observed orientational distribution suggests that it is among the energetically most favorable orientational distributions for binding to antibodies. Thus, the affinity is enhanced by a pre-posed distribution of interdomain orientations while maintaining the flexibility required for function.

The protocol described above can be applied to other biological systems in general. Protein molecule calmodulin and RNA molecule trans-activation response element (TAR) also have intensive interdomain motions with relative small intradomain dynamics. Their interdomain motions were studied using our method based on published RDC data. Our results were consistent with literature results in general. The differences could be due to previous studies' use of physical models, which contain assumptions about potential energy and thus introduced non-experimental information into the interpretations.

Nuclear Magnetic Resonance Spectroscopic and Computational Investigations of Chloroperoxidase Catalyzed Regio- and Enantio-Selective Transformations

Chloroperoxidase (CPO) is the most versatile heme-containing enzyme that catalyzes a broad spectrum of reactions. The remarkable feature of this enzyme is the high regio- and enantio-selectivity exhibited in CPO-catalyzed oxidation reactions. The aim of this dissertation is to elucidate the structural basis for regio- and enantio-selective transformations and investigate the application of CPO in biodegradation of synthetic dyes. To unravel the mechanism of CPO-catalyzed regioselective oxidation of indole, the dissertation explored the structure of CPO-indole complex using paramagnetic relaxation and molecular modeling. The distances between the protons of indole and the heme iron revealed that the pyrrole ring of indole is oriented toward the heme with its 2-H pointing directly at the heme iron. This provides the first experimental and theoretical explanation for the "unexpected" regioselectivity of CPO-catalyzed indole oxidation. Furthermore, the residues including Leu 70, Phe 103, Ile 179, Val 182, Glu 183, and Phe 186 were found essential to the substrate binding to CPO. These results will serve as a lighthouse in guiding the design of CPO mutants with tailor-made activities for biotechnological applications. To understand the origin of the enantioselectivity of CPO-catalyzed oxidation reactions, the interactions of CPO with substrates such as 2-(methylthio)thiophene were investigated by nuclear magnetic resonance spectroscopy (NMR) and computational techniques. In particular, the enantioselectivity is partly explained by the binding orientation of substrates. In third facet of this dissertation, a green and efficient system for degradation of synthetic dyes was developed. Several commercial dyes such as orange G were tested in the CPO-H2O2-Cl- system, where degradation of these dyes was found very efficient. The presence of halide ions and acidic pH were found necessary to the decomposition of dyes. Significantly, the results revealed that this degradation of azo dyes involves a ferric hypochlorite intermediate of CPO (Fe-OCl), compound X.