Master of Science / Department of Kinesiology / Thomas J. Barstow / Near infrared spectroscopy is currently routinely used to assess tissue (muscle) oxygenation at rest and during exercise. While most investigators assume that hemoglobin ([Hb]) is the major contributor to the responses seen during exercise, the relative contribution of myoglobin ([Mb]) to the NIRS signals remains controversial. PURPOSE: a) To calculate the range of light absorbing potential (LAP) of hemoglobin and myoglobin in mammalian skeletal muscle at rest based on analysis of published chemical and morphometric data in humans and other mammals (Part 1), and b) use the information in a) to interpret changes in total [Hb+Mb] from NIRS during exercise (Part 2). METHODS: Part 1: Information was retrieved from five published studies with regard to capillary density (#caps/mm2) and [Mb] in skeletal muscle of human, horse and rat. Preference was given to studies in which both measurements were provided for the same muscles. [Hb] in skeletal muscle was estimated as a function of capillary density, [Hb] in systemic blood, and the ratio of capillary-to-systemic hematocrit at rest and during exercise. Part 2: Changes in total [Hb] + [Mb] (as t[Hb+Mb]) from published NIRS data obtained from human subjects performing cycling or knee extension exercise were interpreted in the context of the results of Part 1. RESULTS: Part 1: Individual group mean values for skeletal muscle [Mb] in the literature ranged from 0.25-0.67 mM in human samples, with a similar range for muscles of the rat hindlimb; horse limb muscles tended to be higher (up to 1.0 mM). Capillary densities ranged from ~200 to 600 caps/mm2 in human and rat muscles, and up to 800 caps/mm2 in horse muscle. Assuming a resting capillary hematocrit of 22% and 4 fold greater LAP for each mole [Hb] vs [Mb], the resulting estimation of capillary [Hb] ranged from ~0.03 to 0.09 mM in human and rat muscles, and up to ~0.13 mM in horse muscles. The results suggest that [Mb] could contribute ~50-70% of the total LAP at rest in human skeletal muscle. Part 2: With exercise, total heme by NIRS can increase ≥ 30% in individual human subjects. Assuming this increase reflects only increased [Hb], this fits well with the observed increase in capillary hematocrit with exercise. CONCLUSIONS: 1) In skeletal muscle at rest, [Mb] is likely to be at least as significant a light absorbing heme as is [Hb] in most mammalian muscles, including the human leg. 2) Observed increases in t[Hb+Mb] with NIRS during exercise can be explained by an increase in capillary hematocrit, even in the presence of significant [Mb].

Doctor of Philosophy / Food Science Institute / Melvin C. Hunt / Several experiments were conducted to evaluate factors affecting myoglobin redox forms stability and detection of myoglobin redox forms using near infrared (NIR) spectroscopy. In experiment 1, we investigated the relationship between metmyoglobin (MMb) reduction and oxidation of malate to α-ketoglutarate with regeneration of reduced nicotinamide adenine dinucleotide (NADH) via malate dehydrogenase (MDH). Our specific objectives for this experiment were: (1) to examine the interaction of malate and MDH to reduce MMb in vitro, (2) to determine the influence of pH, temperature, NAD[superscript]+, and malate concentration on MDH enzyme activity and MMb reduction, and (3) to determine the effects of malate on NADH generation, metmyoglobin reducing activity, and color stability using beef muscles (Longissimus lumborum, Psoas major, and Semitendinosus) extracts. We observed that, nonenzymatic reduction of horse MMb in vitro in a malate-MDH-NADH system increased with increasing NAD[superscript]+ and L-malate concentrations. Our findings further confirmed that reduction of MMb in beef extract was NAD[superscript]+ and malate concentration dependent (p < 0.05). A model system was described for studying mechanisms of enzymatic reduction of metmyoglobin reduction as a means to improve meat color and the results support the hypothesis that malate can replenish NADH via MDH activity, ultimately resulting in stabilizing myoglobin redox chemistry.In experiment 2, we assessed the ability of mitochondrial and cytoplasmic malate dehydrogenase present in postrigor bovine skeletal muscle to utilize malate as fuel for NADH regeneration and MMb reduction via the malate-NAD-MMb system. Furthermore, addition of lactate to beef mitochondrial and cytoplasmic isolates was evaluated to determine if interactions between malate and lactate increased MMb reduction. Addition of malate to isolated beef mitochondrial and cytoplasmic isolates at pH 7.2 increased (p < 0.05) MMb reduction. MMb reduction resulting from the addition of malate and lactate was equal or greater than MMb reduction resulting from malate alone. The findings from this study provided evidence that mitochondria and cytoplasmic proteins isolated from beef skeletal muscles of different metabolic origin differ substantially in their enzymatic composition. Malate-MDH assisted-MMb reduction using Mitochondrial and cytoplasmic isolates from the three beef skeletal muscles exhibited substantial differences in enzymatic compositions and their ability to reduce MMb in vitro. Differences were also observed in the enzymatic characteristics of MDH-assisted-MMb among the three beef muscles. In experiment 3, we investigated the effects of three glycolytic and tricarboxylic acid cycle metabolites on myoglobin redox forms and their in influence on meat color stability. Eighteen combinations of malate (M), lactate (L), and pyruvate (P) were added to beef Longissimus lumborum, Psoas major, and Semitendinosus muscle homogenates to study their effects on metmyoglobin formation during incubation at 25 °C. Changes in surface color at 0, 2, 4, 8, and 12 hrs were evaluated using refecto-spectrophotometry [both L*a*b* and wavelengths specific for MMb]. Results from this study suggests that at 2% concentrations level of the individual metabolites (M, L, or P), the most effective metabolite at retarding MMb formation was L > M > P in the ST, and M > L > P in the PM and LL muscles. MMB was reduced most effectively with combination of metabolites where M+L > M+P > L+P. Enhancement of meat with these metabolites can effectively extend color life of postrigor meat apparently by providing more reducing conditions for myoglobin, thus increasing myoglobin redox form stability. Experiment 4 was conducted to determine how near-infrared (NIR) tissue oximeter measurements of post-rigor beef skeletal muscle relate with the more established methods of quantifying myoglobin redox states. Surface color differences were created by packaging steaks in vacuum (VAC), 80% O[subscript]2 and 20% CO[subscript]2 modified atmosphere packaging (HiOx MAP), polyvinyl chloride film overwrap (PVC), and HiOx MAP converted to PVC (HiOx-PVC) after 2 days. Changes in surface color and sub-surface pigments during display (0,2, 4, 10, and 15 days at 2 °C) were characterized by using a reflectance-spectrophotometer and a near-infrared tissue oximeter, respectively. Fiber orientation, storage, and packaging affected (p < 0.05) color, total pigment, deoxymyoglobin, and oxymyoglobin content. Tissue oximetry measurements appear to have potential for real-time monitoring of myoglobin redox forms and oxygen status of packaged meat, but fiber orientation needs to be controlled. In experiment 5, we investigated the response of frequency-domain multidistance (FDMD) NIR tissue oximetry for detecting absolute amounts of myoglobin (Mb) redox forms and their relationship to meat color stability. Four packaging formats were used to create different blends of Mb redox forms and meat colors during display. Changes in surface color and subsurface pigment forms during simulated display (0, 2, 4, and 10 d at 2 °C) were evaluated using surface reflecto-spectrophotometry (both L*a*b* and specific wavelengths) and FDMD NIR tissue oximetry. Data for both methods of direct measurement of oxymyoglobin and deoxymyoglobin were strongly related and accounted for 86 to 94% of the display variation in meat color. Indirect estimates of metmyoglobin ranged from r[superscript]2 = 59 to 85%. It appears that NIR tissue oximetry has potential as a noninvasive, rapid method for the assessment of meat color traits and may help improve our understanding of meat color chemistry in post-rigor skeletal muscle.

No description available.

<p>There is a critical need to target tumor hypoxia as patients with hypoxic tumors have worse prognosis due to aggressive phenotypes and resistance to radiotherapy and chemotherapy. The overall goal of this work is to improve response to conventional cancer therapies by targeting tumor hypoxia. This has been carried out and evaluated through the use of polymersome-encapsulated myoglobin (PEMs) with the hypothesis that O2-releasing PEMs will increase tumor oxygenation, and thereby improve response to radiotherapy. Mb was chosen as an O2 carrying protein to deliver to tumors because it has a strong association to O2, providing a mechanism to deliver O2 only within the hypoxic regions of the tumor. Mb was loaded within nanoscale polymeric vesicles that were expected to accumulate within solid tumors due to the enhanced permeability and retention (EPR) effect. This hypothesis has been tested through the following aims:</p><p>1. Develop NIR imaging techniques for studying the biodistribution and pharmacokinetics of polymersomes</p><p>2. Establish the effects of Mb-containing polymersomes on tumor physiology</p><p>3. Modify tumor growth through delivery of Mb polymersomes in combination with a cytotoxic therapy specific to aerobic tumors</p><p>These aims have been evaluated through numerous in vivo studies. First, polymersomes of various polymer formulations and diameters ranging from 110-550 nm were prepared with a near-infrared (NIR) -emissive fluorophore. Using live animal fluorescence imaging, I was able to study the biodistribution of the polymersomes following i.v. administration, demonstrating significant polymersome accumulation in orthotopic 4T1 mammary carcinomas. In addition, a novel method for measuring pharmacokinetics was developed, using serial small volume blood draws from individual mice. The plasma fluorescence in microcapillary tubes was used to quantify polymersome concentrations, demonstrating long circulation half-lives that varied from 6-23 h. Toxicity of various polymersome formulations were also studied in vitro and in vivo, revealing negligible toxicities.</p><p>For the second aim, PEMs were administered i.v. in tumor-bearing mice. Unexpectedly, we observed a dramatic gross tumor effect within hours of treatment in both orthotopic 4T1 tumors and flank Renca renal cell carcinomas. Histological analysis revealed endothelial cell apoptosis as early as 1 h following treatment, with scattered tumor cell death throughout the tumor by 4 h. Hematoxylin and eosin staining showed significant necrosis 24 h following PEM treatment. Vascular effects and polymersome distribution were studied in 4T1 window chamber tumors. Following i.v. treatment with PEMs, intravital microscopy was used to image polymersome fluorescence, brightfield transmission was imaged for vessel morphology and blood flow, and a tunable filter was used for determining hemoglobin (Hb) oxygen saturation. Tumor hemorrhaging was observed within hours of PEM treatment, which was not seen with empty polymersomes. This was consistent with the gross tumor effects observed initially. Hb saturation decreased in both the PEM and empty polymersome groups, but not in saline-treated mice. While we expected to observe an increase in tumor oxygenation by using Mb as an oxygen carrier, we actually observed hemorrhage, decreased oxygenation, and central tumor necrosis. In vitro studies using human endothelial cells demonstrated dramatic changes in cell morphology and increased permeability due to Mb and PEM treatments, which appear to be enhanced in an oxidative environment. These in vitro and in vivo observations are similar to what is seen with tumor vascular disrupting agents.</p><p>For the third aim, I combined radiotherapy (RT) and PEM treatment with a new hypothesis. I originally expected the PEMs to increase tumor oxygenation, thus making the tumor more susceptible to RT. However, considering the results from the second aim, this hypothesis was modified: the PEMs would result in necrosis of the tumor core, while RT would target the more oxygenated rim of the tumor, thus leading to improved tumor growth delay compared with PEM or RT alone. This hypothesis was tested in both orthotopic, syngeneic 4T1 tumors as well as flank FaDu xenografts. 4T1 tumor cells were surgically implanted within the dorsal mammary fat pad of mice and grown until ~200 mm3. A CT microirradiator with a square collimator was used in order to locate and specifically irradiate the tumor. Within 1 h following RT, the PEMs were administered i.v.. Mice receiving PEMs with no RT showed a significant decrease in tumor growth compared with saline-treated mice (p = 0.0001 for time to 3x original tumor volume). In addition, the combination of RT plus PEMs reduced tumor growth compared with RT alone (p = 0.0144 for time to 3x original tumor volume). However, this effect was not seen with FaDu tumors. This may have been due to excessive radiation dose or other compounding factors: the timing between RT and PEM treatment was not optimized, and the number of mice per group was small (3-4). </p><p>Thus, the conclusions for each aim are as follows:</p><p>1. Develop NIR imaging techniques for studying the biodistribution and pharmacokinetics of polymersomes</p><p>NIR imaging techniques were optimized for studying polymersomes, demonstrating long plasma circulation times and accumulation within tumors.</p><p>2. Establish the effects of Mb-containing polymersomes on tumor physiology</p><p>While the hypothesis was that PEMs would accumulate within hypoxic tumors and subsequently increase O2 tension, we observed a rapid decrease in tumor oxygenation followed by a dramatic hemorrhagic effect of Mb polymersomes, which appear to be due to both endothelial cell apoptosis and morphological changes, resulting in central tumor necrosis.</p><p>3. Modify tumor growth through delivery of Mb polymersomes in combination with a cytotoxic therapy specific to aerobic tumors</p><p>Combination therapy of PEMs with RT results in enhanced tumor growth delay in aggressive 4T1 mammary carcinomas compared with RT or PEMs alone.</p><p>These studies have led to a proposed mechanism for the PEM anti-tumor effect in combination with RT. Prior to PEM administration, RT is administered, resulting in tumor cell kill of the well-oxygenated tumor periphery. Mb polymersomes are then injected i.v. and begin to accumulate within tumors due to the EPR effect. As shown in Aim 1, this accumulation occurs over a short time scale. Within 30 min of PEM treatment, the Mb is believed to act on tumor vessels, resulting in morphological changes and apoptosis of endothelial cells. These effects are expected to increase permeability of the vessels and expose the basement membrane, which leads to clotting and decreased blood flow. Both decreased perfusion and increased permeability are believed to have a catastrophic effect on interior tumor vessels. Hemorrhage results as the endothelial cells die, resulting in tumor core necrosis. Therefore, the result is tumor cell kill at the periphery due to RT and central tumor necrosis due to PEM treatment.</p><p>PEMs have potential in cancer therapy as a new class of VDAs. While the mechanism requires further investigation, this work has demonstrated that PEM treatment results in tumor vessel destruction and central necrosis. PEMs accumulate within tumors, thus minimizing the systemic toxicity of treatment commonly seen with VDAs. By combining PEMs with a therapy that kills the better perfused tumor periphery, PEMs show promise in improving tumor response. Future mechanistic studies will be needed in order to maximize vessel damage and optimize combination dosing schedules to improve outcome.</p> / Dissertation

As propriedades dos derivados nitrosilados de diferentes hemoproteínas em função da temperatura, da concentração de óxido nítrico (NO) e pH, foram investigadas utilizando a técnica de Ressonância Paramagnética Eletrônica (RPE). Nas hemoproteínas, o grupo heme está acomodado em um bolso: de um lado encontra-se a histidina proximal, de outro uma cavidade aberta onde pode ocorrer a ligação de O2 (ou outras moléculas, tais como H2O, NO, CO, etc.). Perto daí, encontra-se o sítio distal, que pode ou não estar ocupado por uma cadeia lateral de aminoácido, o qual pode influenciar a ligação do ligante. As proteínas estudadas, marcadas com NO, foram: mioglobinas de Cavalo de Cacgalote, que possuem o sítio distal ocupado por um resíduo de histidina, a mioglobina do molusco Aplysia brasiliana (Mb Apb) que não possue um resíduo de aminoácido no sítio distal e a eritrocruorina da minhoca Annelidae Glossoscolex paulistus (Ec AGp), cuja vizinhança do heme não está determinada. Os resultados obtidos com as soluções de mioglobinas de Cavalho e de Cachalote, preparadas a partir da proteína liofilizada (Sigma) mostram cerca de 13% de mioglobina está na forma oxi (MbA) e o restante na forma meta (MbB). A ligação de NO à Mba se faz por simples substituição da molécula de O2 por NO, mantendo o ferro em seu estado ferroso. Já para a MbB, a ligação ocorre em duas etapas: na primeira reduzindo o Fe3+ a Fe2+ e, numa segunda, cuja extensão depende da concentração de NO, o óxido nítrico liga-se ao Fe2+ produto da redução do ferro da MbB. Se a razão NO/Mb é igual ou maior que 2/1, o espectro da RPE é uma linha carga (tipo B), como tem sido identificado na literatura. Se a razão NO/b é da ordem de 0,2/1, o espectro de RPE (tipo A) apresenta um desdobramento hiperfino, similar ao da deoxiHb humana na conformação T. Se a razão NO/Mb varia entre 0,2 e 2, o espectro de RPE é uma mistura de dois tipos (AB). Os espectros tipo A e AB apresentam uma clara dependência com a temperatura, ao passo que o espectro tipo B não. Observa-se que também que a afinidade pelo NO da MbB depende do pH; quanto menor o pH, mais difícil fica se completar a reação do Fe3+ com o NO, mesmo se a razão NO/Mb é maior que 2. Os espectros de RPE da MbANO e da MbBNO são diferentes, porque o campo cristalino em torno do ferro apresenta pequenas variações, alterando a distância de N&#949-FE-NO. No caso da MbA isto dá origem a um espectro similar ao encontrado para o ferro pentacoordenado (tipo A); no caso da MbB, o espectro é do tipo encontrado para ferro hexacoordenado (tipo B). O estudo da dependência do espectro de RPE com a temperatura, no caso da EcNO AGp, mostra que existe um equilíbrio térmico entre duas espécies hexacoordenadas: a espécie I, que predomina à baixa temperatura e cujo espectro de RPE exibe desdobramento superhiperfino com 9 linhas; a espécie II, que predomina altas temperaturas e cujo espectro de RPE é uma linha larga. No intervalo de temperaturas estudado, pode ser observado que em temperaturas intermediárias, os espectros são uma mistura das duas espécies. Pode-se através de diferença espectral estudar o comportamento de cada espécie separadamente e o equilíbrio da mistura. Verificou-se que as duas espécies estão separadas por cerca de 2 kcal/mol. Com os resultados obtidos e comparando-os com os obtidos na literatura, é possível sugerir a existência de uma glutamina distal para a Ec AGP. Para a MbNO ApB, encontra-se um espectro de RE que apresenta desdobramento superhiperfino com 9 linhas. Os espectros a baixa e alta temperatura são similares apenas à alta temperatura e a resolução é menor. Observa-se ainda, a influenciado sítio distal (his E7 nas mioglobinas de Cavalo e de Cachalote) sobre a ligação Fe-N-O. Nas Mb de Cavalo e de Cachalote, em que o sítio distal é ocupado por uma histidina, o espectro da RPE da MbNO é uma linha larga que não varia significativamente com a temperatura. Na Ec AGp, em que o sítio distal está provavelmente ocupado por uma glutamina, o espectro de RPE apresenta uma dependência com a temperatura: a 309.3K, o espectro é uma linha larga, a 103.0K, o espectro apresenta desdobramento superhiperfino. Na Mb Apb, em que não há resíduo básico ocupando a posição distal, o espectro apresenta desdobramento superhiperfino em todo o intervalo de temperatura estudado (113,7K a 283,3K). / Using the Electron Paramagnetic Resonance (EPR) technique, the properties of several nitrosyl hemoproteins were investigated as a function of temperature, pH and nitric oxide (NO) concentration. In hemoproteins, the heme moiety is esconced in a pocket: in one side there is na open cavity where the bonding of different molecules, such as H2O, NO, CO, etc., may occur occupied or not by an aminoacid residue thus influencying in different ways the ligand bonding. The following nitrosyl hemoproteins were studied: horse and spermwhale myoglobins which have an histidine residue occupying the distal site, the myoglobin of the mollusk Aplysia brasiliana (Mb Apb) which has no aminoacid residue in the distal site and the erythrocruorin from the earthworn Annelidae Glossoscolex paulistus (Ec AGp) whose structure is no yet known. The results that were obtained with horse and spermwhale myoglobins solutions prepared using liofilized protein from Sigma, show that almost 13% of the myoglobins is in the oxi form (MbA) and the remaining 87% is in the meta form (MbB). The NO binding to MbA is done by simple substitution of the O2 molecule, remaining the iron atom in its ferrous state. With MbB we have a quite different situation and in this case, two steps are needed to obtain the nitric oxide derivative: first the Fe3+ is reduced to Fe Fe2+ and then the NO molecule bind to it, the extention to which this last step occurs, depends on the nitric oxide concentration. If the NO/Mb ratio is equal to or greater than 2/1, the EPR signal is a broad line (B type), as it has been already identified. If the NO/Mb ratio is around 0,2/1, the EPR spectrum (A type) is characterized by a hyperfine splitting similar to that of human deoxihemoglobin in the T conformation. Finally, in the NO/Mb ratio has a value between 0,2/1 and 2/1, the EPR signal observed is due to contributions from both species (AB). Variations in the EPR spectra with temperature is only observed for the A and AB types. Futhermore, it has also been observed that MbB affinity for NO is pH dependent, the lower the pH, more difficult is the completation of the Fe3+ reaction with NO, even IF the NO/Mb ratio is greater than 2/1. The difference between the EPR spectra of MbA and MbB is due to the small but significative differences in the crystal field around the iron atom, thus modifying the N&#949-FE-NO distance. In the MbA case, this gives rise to a spectrum similar to that find for pentacoordinated iron (A type), for MbB the spectrum shows a pattern similar to that find for a hexacoordinated iron atom (B type). The analysis of the EPR spectra of EcNO AGp at various temperatures showed that there is a thermal equilibrium between two hexacoordinated species: species I, which is predominant at a low temperatures and is characterized by a nine-line superhyperfine splitting, and species II, which is predominant at high temperatures, characterized by a broad line EPR spectrum . It was found that in the range of temperatures studied , the EPR signals are due to contributions from two species. The spectral difference made possible to learn about each species independently and the equilibrium. The two species differ in enthalpy by no more than about 2 kcal/mol. At the light of these findings, and compearing with the data found in the literature, it may be postulated the existence of a glutamine at the distal site in Ec AGp. For MbNO Apb, it was found an EPR spectrum exhibiting a nine-line superhyperfine splitting. The EPR spectra at low and high temperatures are similar, but the former are well resolved while in the latter, the superhyperfine splitting is something blurred. Futhermore, it has been possible to learn about the influence of the distal site on the Fe-N-O bond. For horse and spern whale myoglobins, where na histidine occupies the distal site, the EPR spectrum is a broad line which does not change significatively with temperature. For Ec AGp, where a glutamine probably occupies the distal site, the EPR spectra are temperature dependent: at 309.3K it is a broad line and at 103.0K it presents superhyperfine splitting. Finally, for Mb Apb where there is no basic residue occupying the distal site, the EPR spectra are characterized by a nine-line superhyperfine splitting all over the temperature range studied (113.7K to 283,3K).

A cor vermelha brilhante característica do presunto Parma é resultante, principalmente, do pigmento Zinco-protoporfirina IX (ZnPP). A ZnPP é formada a partir da mioglobina por uma reação de transmetalação, catalisada pela enzima ferroquelatase (FECH), em que o íon de Fe(II) coordenado ao grupo heme é substituído pelo íon Zn(II). O presunto Parma apresenta uma maior estabilidade oxidativa em relação aos demais produtos cárneos curados além de não conter nitrito e nitrato, portanto, são considerados mais saudáveis. A utilização da FECH no processamento de carnes curadas pode permitir a produção de produtos cárneos curados mais saudáveis e em menor tempo. No presente trabalho a proteína ferroquelatase de Bacillus subtilis (BsFECH) foi expressa em células de E. coli BL21(DE3), purificada por cromatografia de afinidade ao níquel e exclusão por tamanho e caracterizada por dicroísmo circular, emissão de fluorescência do triptofano e cromatografia de exclusão por tamanho analítico. Em termos de estabilidade foi encontrado que altas concentrações de sal aumentam a estabilidade da proteína frente aos agentes denaturantes ureia e temperatura. A BsFECH produzida é capaz de ligar-se ao substrato modelo de porfirina (TPPS), conforme verificado por espectroscopia de UV-Vis, com uma Ka = 3,8x105 M-1 e é capaz de se associar à metamioglobina, conforme verificado por reação de cross-linking com dissuccinimidil suberato e avaliado por SDS-PAGE. A BsFECH aumenta significativamente a taxa de inserção de íons de zinco na TPPS e mostra uma cinética de saturação com uma constante de ligação aparente de Zn(II) ao complexo [BsFECH-TPPS] de 1,3x104 M e uma constante de primeira ordem de 6,6x10-1 h-1 para a dissociação do complexo ternário. A reação de troca ferro/zinco na mioglobina catalisada pela BsFECH é facilitada pela proteólise limitada da mioglobina com pepsina que abre um caminho para a reação de troca metálica com base na interação proteína-proteína entre o fragmento globina da mioglobina e a BsFECH. / The bright red color, characteristic of the Parma ham, results mainly of the pigment Zinc-Protoporphyrin IX (ZnPP). The ZnPP is formed from myoglobin by the reaction, catalyzed by ferrochelatase enzyme (FECH), in which Fe(II) ions coordinated to the heme group is replaced by Zn(II) ions. Parma ham shows greater oxidative stability when compared to others cured meat products besides do not contain nitrite and nitrate and, therefore, is considered healthier. The use of FECH in the processing of cured meats may allow the production of healthier cured meat products in a shorter period of time. In this work, the ferrochelatase protein from Bacillus subtilis was expressed in E. coli BL21(DE3) cells, purified by nickel affinity chromatography and size exclusion, and characterized by circular dichroism, fluorescence emission of tryptophan and analytical size exclusion chromatography. In terms of stability, it was found that the high salt content enhances the protein stability against the denaturation agents urea and temperature. The BsFECH produced is able to bind to the porphyrin model substrate (TPPS), as verified by UV-Vis spectroscopy, with Ka = 3.8x105 M-1 and is capable to associate to metamyoglobin as verified by cross-linking reaction with dissuccinimidil suberato, as observed by SDS-PAGE. The BsFECH increases, significantly, the zinc ions insertion rate in TPPS and shows a saturation kinetics behavior with an apparent biding constant of Zn(II) to the [BsFECH-TPPS] complex of 1.3x104 M and a first order rate constant for the dissociation of ternary complex of 6.6x10-1 h-1. The Fe/Zn exchange reaction in the myoglobin as catalyzed by BsFECH is facilitated by myoglobin-limited proteolysis with pepsin that opens a reaction channel for the metallic exchange based on protein-protein interaction between the globin moiety of myoglobin and BsFECH.

Due to the large gaps in the fossil record, the evolutionary history of the mammalian order Cetacea is incomplete and controversial. Increasingly researchers are utilizing molecular and biochemical procedures to supplement cetacean paleontology. One of these methods is the comparison of amino acid sequences of myoglobin among species of this order. since this method is time-consuming and expensive, an alternative procedure is desirable.

Myoglobin (Mb) oxidation, and the subsequent browning, is the primary basis for consumer rejection of fresh retail beef. Considerable effort has been directed by the industry towards the development of techniques that can enhance color stability. However, the underlying mechanism of Mb oxidation has been studied extensively, but is still not entirely understood. It is known that chelation of iron and copper delays Mb oxidation and browning, but a clear role for these metals has not been established in any current Mb oxidation mechanism. The objective of the current study was to examine the possibility that iron plays a more direct role in Mb oxidation, and that metal chelators such as milk mineral (MM) and sodium tripolyphosphate can inhibit this action. MM, a colloidal calcium phosphate of large molecular weight and undetermined structure, was demonstrated to be a high-affinity iron chelator. Non-heme iron was found to stimulate Mb oxidation even in the absence of lipid, showing for the first time that the role of ferrous iron was not limited to promoting lipid oxidation, but instead has a yet-to-be determined role as a pro-oxidant factor in Mb oxidation. Ferrous iron was found to promote Mb oxidation under standard atmospheric conditions, while in high oxygen systems this effect was not seen. Addition of catalase did not affect Mb oxidation. However, in iron-containing systems, catalase significantly slowed Mb oxidation, while MM addition completely reversed the stimulatory effect of added iron. Type I radical-quenching antioxidants were found to rapidly reduce ferric iron to the ferrous form. This strong reducing ability accounted for the pro-oxidant effects of rosmarinic acid and eugenol in the lipid-free Mb model system. In raw ground beef, Type I antioxidants were highly effective at preventing Mb oxidation in the presence of lipid. Of the Type II chelators examined, only MM was able to delay Mb oxidation as well as the Type I antioxidants, possibly because it is not as susceptible to enzymatic hydrolysis.

Although nitrite is a known inhibitor of Clostridium botulinum in cured meats, the mechanism of inhibition is not understood. The observation has been made that iron is required for growth of C. botulinum and that the role of nitrite may be to alter the pathway of iron uptake by these organisms. Since the color change in cured meats is due to the binding of nitrite to the heme group of meat pigments, it was hypothesized that nitrite may also be tying up an essential iron source, heme. This experiment was an investigation of the possibility that myoglobin added to a meat system would stimulate growth and toxin production by C. botulinum much more than myoglobin that had been nitrosylated before inclusion in the product. Treatments were included to compare the effects of a heme iron source, myoglobin, with that of an ionic source, ferric chloride. To help understand the role of free iron in botulinal growth, several treatments contained a metal ion chlator, ethylenediaminetetraacetic acid (EDTA). Nitrite caused a definite delay of growth, as evidenced by gas bubbles, when compared with a non-nitrite system. Addition of ferric chloride resulted in an increase in the rate of of appearance of swollen samples, although growth was enhanced even more when myoglobin was added. When nitrosylated myoglobin was included, growth was inhibited more than in the treatment with nitrite alone. EDTA inhibited growth of C. botulinum but a conclusion should not be made with respect to the chelation of iron since EDTA chelates many other metals. Residual nitrite levels had declined to below 10 ppm by the time swelling occurred. Although swelling did not occur until nitrite had declined in the products, the absence of nitrite alone did not allow growth and toxin production. Since nitrosylated myoglobin and EDTA inhibited botulinal growth even after residual nitrite had declined, it is possible that the inhibitory action of nitrite is creating a nutritional deficiency for C. botulinum.

Proteins have evolved over millions of years to serve a plethora of highly specialized functions in biological systems. Given the enormous diversity in structure and function, it is truly surprising that only 20 different amino acids are utilized as the building blocks of proteins. Furthermore, only a small set of metal cations that are biologically available are used as structural or catalytically active cofactors in proteins, whereas rare metal cations such as platinum, ruthenium or rhodium remain absent. In the 20th century myriad catalysts, based on non-biological transition metals, emerged that can facilitate numerous organic transformations. The goal of the thesis was to introduce new functions into proteins by attaching platinum metals and fluorescent metal sensors. Thus, semi-synthetic proteins for catalytic and analytical applications were generated. The replacement of organic solvents by environmentally benign solvents such as water is an imperative step towards achieving "green chemistry". The combination of small molecule catalysts with proteins may introduce new functions and take advantage of the benefits of "both worlds" while avoiding their potential drawbacks. Therefore semi-synthetic catalysts were developed for enantioselective organic reactions in aqueous medium. A suitable reaction, reaction conditions and catalytic system for later utilization in a semi-synthetic protein were designed, developed and characterized. Ruthenium porphyrins catalyzed cyclopropanation reactions with fair yields and high stereoselectivity in aqueous medium. The successful reaction in water was a crucial requirement for a catalytically active semi-synthetic protein. Mechanistic studies did not elucidate the actual catalytic species for the formation of the cyclopropanation product and the side-product diethyl maleate; however, new insights were gained from the analysis of potential reaction pathways. Moreover, studies of the influence of axial ligands, resembling likely residues coordinating to the ruthenium metal center in the active site of a semi-synthetic protein, on the carbene formation of ruthenium porphyrins illustrated that coordination of axial ligands may inhibit the catalytic activity. The generation of ruthenium porphyrin based semi-synthetic proteins and their subsequent catalysis of cyclopropanation reactions was carried out. Myoglobin and myoglobin mutants were successfully reconstituted with a heme-like ruthenium carbonyl porphyrin; however, none of the formed semi-synthetic proteins catalyzed the enantioselective cyclopropanation of styrene. Efforts to determine the reconstitution efficiency of the generated semi-synthetic were hampered by problems to purify the generated semi-synthetic proteins that are probably due to non-specific binding of the ruthenium porphyrin to the protein surface. The exploration of labile metal pools of the biologically relevant transition metals copper, iron and zinc in cells was the goal of developing semi-synthetic proteins for analytical applications. Combining fluorescent proteins with colored or fluorescent metal chelators by forming semi-synthetic proteins allows taking advantage of their beneficial properties while avoiding their downsides. This design offers an attractive platform for in vivo metal sensing. Plasmids encoding fluorescent proteins, targeting sequences and AGT or intein fusion domains (necessary for labeling) for eukaryotic and prokaryotic expression were generated. The targeting of intracellular compartments (mitochondria, nucleus and TGN) was successful (confirmed by light microscopy experiments with transfected mammalian cells). In vitro labeling experiments of expressed and purified fusion proteins with rhodamine derivatives succeeded with AGT based fusion proteins; however, labeling of fusion proteins by trans-splicing with split-inteins failed. A new Zinc(II)-chelator was attached to an AGT based protein and the resulting semi-synthetic protein exhibited strong changes of fluorescence in the presence of zinc(II). This represents an important step towards the goal of in vivo cell imaging of labile zinc(II) pools. Despite extensive efforts, all attempts failed to generate a chelator that forms Cu(I)-complexes with the 1:1 stochiometry (ligand:metal) that is necessary for metal sensing with semi-synthetic proteins.