This work focuses on pigmy sperm whale and horse myoglobins (Mbs), which are distinguished by a single heme pocket residue variant in the CD3 position, when the heme iron is in the +3 oxidation state (i.e. the met form). The strategy employed is as follows: (i) assign heme peripheral protons; (ii) assign the amino acid residues from the heme cavity; (iii) assess the dynamics of ligand binding in the active site by means of hydrogen Iability, solvent isotope effects, and heme-insertion isomer trapping, all by NMR methods. The results of these studies portray dynamic solution structure of the Mb ligand binding site, and provide a set of standard parameters for the studies of larger hemoproteins. The findings are also important for understanding protein-ligand interactions in general. My research investigates the mixed spin metazido and metimidazole complexes of Mbs for the following reasons. First, the allosteric properties of hemoglobin arise mainly from the transition between its two possible quaternary structures. This can be studied by paramagnetic NMR because it is one of the most sensitive tools in terms of changes in the molecular and/or electronic structure of the heme. Second, both the N₃- and imidazole (lm-) complexes are good compromises, in terms of sizes, between the small diatomic oxygen or CN⁻ molecules and the bulky phenyl group. Thus, we can determine the influence of ligand size on structural perturbation of the Heme crevice by comparison among the different size groups. Third, the saturation-transfer phenomenon between metMbIm and metMbH₂0 provides a route to assignments in metMbH₂0 by using assignments of metMbIm. This is crucial because metMbH₂0 is the basis of theoretical calculations of the isotropic shift due to axial ligand field in pure high-spin hemoproteins. Finally, the importance of the metMbIm is underscored by the fact that it is a bis-imidazolium complex, which can then serve as a model other bis-histidyl proteins. Most of the heme peripheral resonances of metEqMbIm and metEqMbN₃ were identified by means of two-dimensional NOESY,COSY, and EXSY spectroscopy. The strongly relaxed upfield protons in metMbIm were assigned based on steady-state 1D NOE and T₁ experiments. Based on the results from metMblm in which saturation transfer of one upfield resonance led to two different free ligand peaks, bound Im equilibration was envisioned and proven by the divergence of broad downfield heme methyl peaks into two peaks each, showing distinctive population preference of each isomer. Dicyanoheme probe, as well as hydrogen Iability comparison studies between pigmy sperm whale Mb and horse Mb in the azido and imidazole states, asserts that single variant pocket residue CD3 is crucial in gating the ligand mobility into and out of the active site. The assignments of heme peripheral and upfield resonances enabled the subsequent assignments of some heme pocket amino acid residues. The facile exchange of bound Im with solvent H₂0 lays the ground work for identification of heme pocket residues in metMbH₂0. Furthermore, while deuterated heme previously allowed only assignment of the non-diastereomeric specific heme 2-vinyl β proton, saturation-transfer from horse imidazole Mb affords the specific identification of 2Hᵦt.

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O pigmento da carne fresca, oximioglobina, e a sua forma oxidada, metmioglobina, podem ser ambos oxidados pela riboflavina quando expostos à radiação luminosa, afetando sua estabilidade redox da carne do ponto de vista nutricional e sensorial. A reação da MbFe(II)O2 e da MbFe(III) com o estado tripleto da riboflavina, 3Rib, envolve uma eficiente transferência de elétrons entre o anel isoaloxazina da riboflavina e a cadeia polipeptídica da proteína, o que leva à formação de cross-link e/ou fragmentação, como demostrado por SDS-PAGE e Western-blot. A constante global de velocidade para a oxidação da MbFe(II)O2 pela 3Rib é (3,0 ± 0,5 ) 109 L·mol-1·s-1 e de (3,1 ± 0,4) 109 L·mol-1·s-1 para a oxidação da MbFe(III) pelo estado tripleto da riboflavina. Cálculos termodinâmicos demonstram ainda que há formação de um complexo exotérmico com estequiometria 1:1 favorecido a temperaturas mais baixas com Ka = (1.2 ± 0.2) 104 mol·L-1 a 25 °C e ΔHo = -112 ± 22 kJ·mol-1 e ΔSo = -296 ± 75 J·mol-1·K-1. Conclui-se que para carne, a riboflavina é um fotossensibilizador para oxidação de proteína e não para a descoloração. / The fresh meat pigment oxymyoglobin, MbFe(II)O2, and its oxidized form metmyoglobin, MbFe(III), are both oxidized by riboflavin as photosensitizer. The reaction of MbFe(II)O2 and MbFe(III) with triplet-state riboflavin, 3Rib, involves the pigment protein, which is oxidatively cleaved or dimerized as shown by SDS-PAGE and Western-blotting, while the heme iron center is not oxidized. The over-all rate constant for oxidation of MbFe(II)O2 by 3Rib is (3.0 ± 0.5) 109 L·mol-1·s-1 and (3.1 ± 0.4) 109 L·mol-1·s-1 for MbFe(III) in aqueous 0.20 mol·L-1 NaCl phosphate buffer of pH 7.4 at 25 °C as determined by transient absorption laser flash photolysis. The high rates are rationalized by ground state hydrophobic interactions as detected as static quenching of fluorescence from singlet-excited state riboflavin by myoglobins using single photon counting time resolved fluorescence spectroscopy and a Stern-Volmer approach. Binding of riboflavin to MbFe(III) has Ka = (1.2 ± 0.2) 104 mol·L-1 at 25 °C with ΔHo = -112 ± 22 kJ·mol-1 and ΔSo = -296 ± 75 J·mol-1·K-1. For meat, riboflavin is concluded to be a photosensitizer for protein oxidation but not for discoloration.

The purpose of this study was to assess factors that can influence the colour stability of fresh sausage products using a pork patty model system over a typical distribution and display period. Fresh sausage is usually sold in raw; and it should have minimum 7.5% meat protein and 9% total protein. Losses of meat quality were evidenced through the discolouration of meat, depletion of endogenous antioxidant activities, proliferation of spoilage microorganisms, and reduction in the meat redox potential. <p>Both ground pork and fresh pork patties were made from pork picnic shoulder. In the first study, the quality of both ground pork patties and fresh pork sausage patties decreased over time during storage at 4°C. The fresh sausages contained ingredients that could prolong their shelf life. The activities of these antioxidant enzymes in both ground pork and fresh sausage were depleted by day 5 of the display period. Ground pork, however, had significantly (p<0.05) higher activity of catalase, glutathione peroxidase (GSHPx), superoxide dismutase (SOD) and total antioxidant activity (TEAC) than fresh sausage due to the effect of the salt. Moreover, there was no significant treatment effect on microbial numbers but there was a significant (p<0.05) elevation of microbial colony forming units by day 5 of the display period. The elevation of microbial numbers by the end of the display period was consistent with the drop in redox potential that was measured near the surface of the patties at the end of the incubation period. <p>In the second study, there was no synergistic effect (p<0.05) between sodium erythorbate and lemon juice powder that were used to enhance colour stability during storage and display in terms of antioxidant activity, colour and microbiological profile. The addition of sodium erythorbate alone, however, had a significant effect (p<0.05) on catalase activity and a* values. In other words, this catalase activity was more effective in protecting against oxidation with the addition of sodium erythorbate so that the redness of the fresh sausages (a*values) was preserved. Furthermore, the combined addition of sodium erythorbate and lemon juice powder did not have any antimicrobial activity because there was no significant difference in total microbial counts (Brochothrix thermosphacta count and lactic acid bacteria) following the addition of those ingredients. The measurement of redox potential near the surface of fresh pork patties could not be conclusively correlated with the addition of non-meat ingredients or microbiological activity. However, the measurement of redox potential in the middle of fresh pork patties showed that the addition of sodium erythorbate lowered the redox of the fresh sausage B (0.05% sodium erythorbate) and D (0.25% lemon juice powder and 0.05% sodium erythorbate) compared to fresh sausage A (0.00% lemon juice powder and 0.00% sodium erythorbate).

The purpose of this study was to assess factors that can influence the colour stability of fresh sausage products using a pork patty model system over a typical distribution and display period. Fresh sausage is usually sold in raw; and it should have minimum 7.5% meat protein and 9% total protein. Losses of meat quality were evidenced through the discolouration of meat, depletion of endogenous antioxidant activities, proliferation of spoilage microorganisms, and reduction in the meat redox potential. <p>Both ground pork and fresh pork patties were made from pork picnic shoulder. In the first study, the quality of both ground pork patties and fresh pork sausage patties decreased over time during storage at 4°C. The fresh sausages contained ingredients that could prolong their shelf life. The activities of these antioxidant enzymes in both ground pork and fresh sausage were depleted by day 5 of the display period. Ground pork, however, had significantly (p<0.05) higher activity of catalase, glutathione peroxidase (GSHPx), superoxide dismutase (SOD) and total antioxidant activity (TEAC) than fresh sausage due to the effect of the salt. Moreover, there was no significant treatment effect on microbial numbers but there was a significant (p<0.05) elevation of microbial colony forming units by day 5 of the display period. The elevation of microbial numbers by the end of the display period was consistent with the drop in redox potential that was measured near the surface of the patties at the end of the incubation period. <p>In the second study, there was no synergistic effect (p<0.05) between sodium erythorbate and lemon juice powder that were used to enhance colour stability during storage and display in terms of antioxidant activity, colour and microbiological profile. The addition of sodium erythorbate alone, however, had a significant effect (p<0.05) on catalase activity and a* values. In other words, this catalase activity was more effective in protecting against oxidation with the addition of sodium erythorbate so that the redness of the fresh sausages (a*values) was preserved. Furthermore, the combined addition of sodium erythorbate and lemon juice powder did not have any antimicrobial activity because there was no significant difference in total microbial counts (Brochothrix thermosphacta count and lactic acid bacteria) following the addition of those ingredients. The measurement of redox potential near the surface of fresh pork patties could not be conclusively correlated with the addition of non-meat ingredients or microbiological activity. However, the measurement of redox potential in the middle of fresh pork patties showed that the addition of sodium erythorbate lowered the redox of the fresh sausage B (0.05% sodium erythorbate) and D (0.25% lemon juice powder and 0.05% sodium erythorbate) compared to fresh sausage A (0.00% lemon juice powder and 0.00% sodium erythorbate).

The objective was to examine the use of acetic acid (AA) with xanthan gum (XG) or konjac flour (KF) to reduce variation in cooked color of ground beef patties varying in myoglobin and pH. Beef clods were selected from carcasses of young (<24 months, Y) and mature (>48 months, M) animals. Within each age category, high (>6.0, H) and normal pH (5.3-5.7, N) clods were chosen. Ground beef was prepared from each maturity/pH combination and treatments applied at 12% of the meat block: control (mixed only), 0.5% AA, 0.25% XG/0.5% AA, or 0.125% KF/0.5% AA. Dry and moist cooking was performed in a convection oven to internal temperatures: 65.6 degrees C, 71.1 degrees C, and 76.7 degrees C. Patties were held at 76.7 degrees C for up to 240 min in dry and moist environments. Internal (assessed at 0, 120, and 240 min of holding) and external (assessed every 30 min, 0 to 240 min of holding) color evaluations (CIE L*a*b*, visual doneness, and pink scores) were conducted. Three replications were performed. The YN patties had the most done appearance internally and the highest denatured myoglobin percentage. Generally, the YH and MN patties had responses between YN and MH got most variables. The MH patties had the highest internal a* color space values, lowest degree of doneness scores and low percentage of denatured myoglobin. The YN patties responded normally to the different internal temperatures achieved during cooking. The YH, MN, and MH patties had increased doneness to 71.1 degrees C and plateaued between 71.1 degrees C to 76.7 degrees C. Visual degree of doneness decreased during moist holding and this was most evident in dry cook/moist held patties. Patties from MH meat were not affected by the treatments as much as the other meat types. The inclusion of AA, XG/AA, and KF/AA in patties made from YH and MN can effectively reduce visible redness and increase myoglobin denaturation in comparison to the control YN beef patties. These ingredients could be viable options to reduce the variation that pH or myoglobin content imparts on ground beef patty cooked color, but as seen in the MH meat, treatment additions were not effective for overcoming both pH and high myoglobin content.

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Graduation date: 1961