Progress 04/01/19 to 10/31/23
Outputs
Target Audience:The target audience includes individuals working in the following fields: meat science, electrical engineering, biochemistry, mass spectroscopy, food science, protein crystallography, biophysical chemistry, seafood technology, aquaculture, and the meat industry. Specific organizations with interest include the American Meat Science Association (AMSA), Institute of Electrical and Electronics Engineers (IEEE), American Society for Mass Spectrometry (ASMS), Institute of Food Technologists (IFT), American Oil Chemists Society (AOCS), Poultry Science Association (PSA), Midwest Advanced Food Manufacturing Association (MAFMA), National Pork Producers Council (NPPC), North American Meat Institute (NAMI), National Cattlemen's Beef Association (NCBA), and the Trans-Atlantic Fisheries Association (TAFT). Knowledge from this project was also delivered to graduate students via two graduate level courses: The Chemistry of the Food Lipids 710 and Food Biochemistry 711 that are taught on the University of Wisconsin-Madison campus. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Threegraduate students worked on this project. All three have graduated and received a unique experience by traversing the fields of electrical engineering, analytical biochemistry, and food chemistry. Advanced mass spectrometrystudies were part of this project and sophisticated software wasutilized to interpret the mass spec data. One of the graduate students attended a 3-daymass spectrometry workshop as part of their training. How have the results been disseminated to communities of interest?The findings from this project have been presented at scientific conferencesand in peer-reviewed journal articles. Additional manuscripts from the project are to be submitted for peer review in the coming months of this year. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This work has led to improved understanding of the mechanisms by which discoloration and off-flavor occur in muscle foods during storage. This should lead to better antioxidant strategies as other researchers become familiar with the nuanced nature by which hemoglobin (Hb) and myoglobin (Mb) cause discoloration and off-flavor formation, shortening shelf-life and leading to food waste-a change in knowledge has taken place from this work. Our work examined two important ways in which quality deterioration occurs in raw muscle. The first was discoloration of raw muscle that involves reactions in which the red color of oxyMb and oxyHb is converted to the brown color of metMb and metHb. The second was fat oxidation, a process in which off-odor and off-flavor formation occurs during storage as the fats of the muscle undergo oxidative deterioration. We went into this project with the hypothesis that solvent entering the upper (distal) heme crevice leads to the discoloration reactions (e.g. oxyMb to metMb) while solvent entering the lower (proximal) heme crevice dissociates the hemin moiety from metHb to incur off-flavor formation. The dissociated hemin readily oxidizes the fat component of the muscle. It is highly challenging to measure solvent entry into the heme crevices of Hb and Mb so we teamed with electrical engineers and analytical biochemists to use an approach called plasma-induced modification of biomolecules (PLIMB) in combination with tandem mass spectrometry (MS/MS). PLIMB causes modifications to solvent accessible amino acids of the protein and MS/MS quantifies the degree of modification. PLIMB generates oxygen-containing free radical species from dielectric barrier discharge to covalently modify solvent accessible sites. A refined concept that evolved was that a hydronium ion in the solvent protonates a specific amino acid of the heme crevice in Hb and Mb to start the cascade of reactions that lead to discoloration and off-flavor during storage. PLIMB-MS/MS is somewhat unique in that it has the capacity to measure solvent accessibility at the resolution of a single amino acid of the protein. Other analytical techniques to measure solvent access into the interior of proteins have the shortcoming of not having resolution to a single amino acid in the protein. Even the nuclear magnetic resonance (NMR) approach to measure protonation events at the resolution of a single amino acid is not possible for Hb and Mb due to unavoidable changes in the heme proteins during the process of data acquisition to determine the protonation status. In other words, to measure the discoloration pathway, the oxyHb and oxyMb need to remain as oxyHb and oxyMb during data acquisition. And to measure hemin dissociation, the metHb and metMb need to remain as metHb and metMb during data acquisition. This further emphasizes that PLIMB is a valued approach to explore solvent access to the heme pocket of Hb and Mb. At the same time, the plasma exposure time must be limited to a timeframe in which oxyHb and oxyMb are maintained for studying the discoloration pathway while metHb must be maintained for studying the hemin dissociation pathway. The native protein structure must also be maintained. We determined the maximum amount of time of plasma exposure before loss of i) native structure and ii) desired form of each heme protein using circular dichroism and optical spectroscopy, respectively. Regarding hemin dissociation pathway, we compared two metHbs with widely varying hemin affinity. The heme proteins were trout IV metHb and bovine metHb representing fish and beef, respectively. The fish metHb released its heme moiety around 20-fold faster than bovine metHb which provided a good scenario to explore the relationship between solvent access to the heme pocket and hemin dissociation rate. It was originally thought there would be evidence of greater solvent accessibility to the lower heme crevice in the fish metHb to better understand the pathway by which hemin dissociation occurs. This was not observed so that our hypothesis that a protonation event in the lower heme pocket facilitates hemin loss was not demonstrated. We came to understand that some aspects of the MS/MS methodology used limited quantifying solvent access to certain parts of the fish metHb heme crevice. We now have developed an alternative MS/MS approach that will be able to advance the field of measuring solvent access to the heme crevice as part of the pathway by which hemin dissociates from metHbs to incur off-flavor formation. Further, we have utilized recent advances in computational approaches to estimate protonation tendencies of sites in the heme crevice that provides a focal point for further exploration of the precise mechanism by which hemin release from metHbs occurs. The PLIMB-MS/MS approach did demonstrate that there was more solvent accessibility to the trout heme crevice at site CE3 whereas there was more solvent access to bovine metHb at site C7. This demonstrated PLIMB can be used to examine some aspects of solvent access to different heme crevices. Further, existing structures of each metHb provided mechanistic reasoning that explained the differing solvent accessibilities observed. We also explored if covalent binding of a lipid oxidation product to oxyMb increased solvent access to the heme crevice in the context of converting oxyMb to metMb. 4-Hydroxy-2-nonenal (HNE) is an unsaturated aldehyde that forms during lipid oxidation in muscle foods. Previous work has demonstrated that Mb reacted with HNE undergoes more rapid metMb formation compared to Mb absent of HNE. The mechanism by which the metMb formation is increased in the presence of HNE is incompletely understood. More heme crevice modifications due to PLIMB were observed in HNE adducted oxyMb compared to unadducted oxyMb when collectively evaluating three of the modified sites (P <0.05). One of the sites was the protonatable HisE7 supporting the concept that a hydronium ion in the solvent protonates HisE7 of the heme crevice in oxyMb to start the cascade of reactions that lead to metMb formation during storage. The mechanism by which oxyMb is converted to metMb was further explored by measuring modifications to the heme crevice at pH 6.0 compared to pH 7.8, noting that metMb formation occurs more rapidly at decreased pH. Two techniques were used to extend the plasma exposure time before metMb formation occurred so that solvent access could be better evaluated. One was addition of catalase to remove hydrogen peroxide and the other was replacing the air atmosphere with argon. The determined solvent accessibility of His64 is consistent with the postulated mechanism that protonation of His64 facilitates metMb formation. His64 was not more modified at the lower pH. This suggested access of hydronium ion to His64 was similar at both pH values so that rapid discoloration of muscle at lower pH values is due to the greater abundance of hydronium ions at lower pH, rather than an increase access of those ions to His64 at lower pH. We also explored solvent access to trout IV Hb at pH 6.5 compared to pH 7.2 noting that deoxyHb content is elevated at the lower pH. One non-protonatable site of the heme crevice, αPhe(G5), was more modified at the higher pH. This is consistent with less solvent access to deoxyHb compared to oxyHb which supports a mechanism of metHb formation that involves O2 on the periphery of the heme crevice removing an electron from the heme iron of deoxyHb to incur metHb formation, rather than solvent entry into the heme crevice or a key protonation event in the heme crevice facilitating metHb formation. This work improved our understanding of discoloration and off-flavor formation. The foundation is in place to implement novel antioxidant strategies based on our findings. Further, the precise mechanisms involved can now be further elucidated which should lead to other antioxidant technologies in muscle foods.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2022
Citation:
Whalin, James. PhD Dissertation. Hemoglobin and Myoglobin: An Investigation of the Mechanism of Autooxidation and Heme Affinity using Plasma-Induced Modification of Biomolecules (PLIMB). University of Wisconsin-Madison
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2023
Citation:
Wu, Yuting. PhD Dissertation. Measurements of the Residue-Level Oxidation of Myoglobin Using Plasma-Induced Modification of Biomolecules and Mass Spectrometry. University of Wisconsin-Madison
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2021
Citation:
Blatz, Joshua. PhD Dissertation. The Effects of Electrical, Physical, and Chemical Parameters on a Dielectric Barrier Discharge with a Liquid Electrode. University of Wisconsin-Madison. 2021
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
JM Blatz, D Benjamin, FA Choudhury, BB. Minkoff, MR Sussman, JL Shohet. Effect of frequency and applied voltage of an atmospheric-pressure dielectric-barrier discharge on breakdown and hydroxyl radical generation with a liquid electrode. J. Vac. Sci. Technol. A 38, 043001 (2020); https://doi.org/10.1116/6.0000125
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Progress 04/01/22 to 03/31/23
Outputs
Target Audience:The target audience includes individuals working in the following fields: meat science, electrical engineering, biochemistry, mass spectroscopy, food science, protein crystallography, biophysical chemistry, seafood technology, aquaculture, and the meat industry. Specific organizations with interest include the American Meat Science Association (AMSA), Institute of Electrical and Electronics Engineers (IEEE), American Society for Mass Spectrometry (ASMS), Institute of Food Technologists (IFT), American Oil Chemists Society (AOCS), Poultry Science Association (PSA),National Pork Producers Council (NPPC), North American Meat Institute (NAMI), National Cattlemen's Beef Association (NCBA), and the Trans-Atlantic Fisheries Association (TAFT). Two presentations (one oral and one poster) were given at theAMSA annual meeting in 2022; the oral presentation was for a Symposium session having well over one hundred attendeesA presentation has been submitted to the IEEE audience for the annual Plasma Science meeting in 2023. A presentation has been given to AOCS audience in 2023.Three publications have been published in peer-reviewed journals in the areas of food chemistry and meat science. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two graduate students worked on the grant during this reporting period. One has graduated (PhD) and another graduate student is soon to graduate with a PhD. This grantis a unique opportunity for cross-discipline work in fields encompassing agriculture, biochemistry, and electrical engineering. Advanced mass spectroscopy studies are part of this project, and a user-friendly software is being utilized to interpret the mass spec data. Another graduate student using the same PLIMB technology but for a separate projecthas interfaced with the graduate students of our grant,and a modified approach will be used in our grant to better evaluate solvent access to myoglobin. How have the results been disseminated to communities of interest?The current findings have been presented at scientific meetings (four presentations at three separate conferencesin this reporting period) and in peer reviewed journals (three articlesin this reporting period). What do you plan to do during the next reporting period to accomplish the goals?One more trial is planned to evaluate the effect of mixing during plasma exposureon solvent access measurements. It is envisioned that more of the protein sample will come into contact with the hydroxyl radicals generated by the plasma due to mixing. In other words, results will be obtained that are more representative of the entire protein sample rather than only measuring solventaccessof theprotein molecules that are at theair-liquid interface. This should provide more accurate measurement of solvent access to the heme crevice of myoglobin.
Impacts
What was accomplished under these goals?
IMPACT:This project addresses the problem of food waste and loss of revenue in relation to foods that contain myoglobin and hemoglobin. Discoloration in beef alone causes 1 billion dollars of lost revenue annually in the United States. Such foodsincludemeat, fish, poultry and meat substitutes containing added heme protein. The muscle food and meat substitute sectors will be most impacted by this project. Regarding Goal 1, the mechanism ofmetmyglobin (metMb)formation was examined in this reporting period. MetMb formation is indicative of the red to brown discoloration that occurs in raw meat during storage. Our approach was to modify solvent accessible sites of myoglobin by a technique called plasma-induced modification of biomolecules (PLIMB). Then quantify the modifications due to PLIMB by mass spectrometry. Our primary hypothesis is that one site of Mb, the distal histidine (His-64), is solvent accessible which facilitates metMb formation. If this can be demonstrated, antioxidant strategies can be envisioned by which to inhibit metMb formation. A major activitycompleted wasto collectively examine findings from six separate trials and generate conclusions to better understand the mechanism of metMb formation. We focused our attention on sites that were found to be increasingly modified with increased plasma exposure time in all six trials. This provides the most robust findings. It turned out that the distal histidine was increasingly modified with increasing exposure time in all six trials. Thus, a key accomplishment realized is that our experimentsprovide ample evidence that solvent access to the distal histidine is likely part of the mechanism by which metMb formation occurs. We can now envision targeted strategies to inhibit discoloration in myoglobin-containing foodswith this in mind.An additional hypothesis was that solvent enters into the interior of the heme crevice to facilitate metMb formation. This can be investigated by determining if any internal heme crevice sites were found to be increasingly modified with increased plasma exposure time. Two interior heme crevice sites were found to be increasingly modified with increased exposure time but only in 3 of 6 trials for the interior located isoleucine-107 and 4 of 6 trials for the interior located methionine-142. Thus, this second hypothesis is supported but a definitive conclusion remains elusive. Thus, another accomplishment realized is that our findings provide a justification for developing novel strategies to limit solvent access to the heme crevice of myoglobin to inhibit metMb formation associated with food waste.Once the sites found to be solvent accessible were determined using analysis of variance, a t-test was done to determine if acidic conditions increase solvent access to the heme crevice. Thirteen assessments were done in this regard (six at His-64, four at Met-142 and three at isoleucine-107). Four of the 13 assessments (31%) indicated statistically more solvent access at the lower pH value. None of the 13 assessments (0%) indicated statistically increased solvent access at the higher pH value. This provides trending data of a structural change that occurs at acidic conditions increasing solvent access to the heme crevice to facilitate metMb formation. Thus, a key accomplishment realized is that our approach provides insight regarding the mechanism by which low pH may accelerate metMb formation. Future studies can further establish this finding and potentially lead to novel antioxidant strategies to limit food waste and lost revenue. Off-flavor formation in muscle foods due to deterioration of the fat component is another contributor to food waste and economic losses. Regarding Goal 4, a detailed analysis of solvent access to the heme crevice of bovine hemoglobin (Hb) to that of trout Hb has been conducted. Fish muscle is more prone to fat deterioration compared to beef. Part of this may be due to a greater propensity of fish Hb to deteriorate fat compared to bovine Hb. Our hypothesis was that solvent access to the heme crevice of the fish Hb is greater providing a better understanding of the mechanism by which off flavor occurs in muscle foods. Solvent entering the heme crevice provides hydronium ions that can release the heme moiety from the protein portion of Hb so that the released heme deteriorates the fat through an oxidative process. Our approach was to modify solvent accessible sites of each Hb by PLIMB, and then quantify the modifications due to PLIMB by mass spectrometry. In short, our results did not indicate that the fish Hb heme crevice was more solvent accessible than the bovine Hb heme crevice. At the same time, our work led to surmising there was a highly modifiable amino acid adjacent to the critical proximal histidine in one of the fish Hb chains that limited measurement of solvent access; so that an alternative analytical approach is envisioned to better investigate solvent access to the fish Hb heme crevice. We are currently assimilating recent literature to provide complementary and alternative explanations for the rapid heme release that occurs from fish hemoglobin. Thus, a key accomplishment realized is that our experimentsprovide foundational knowledge to better understand the mechanism by which heme loss occurs from hemoglobin. These findings related to Goal 4 can be used to develop novel antioxidant strategies to better stabilize fats in muscle foods.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
J Whalin, Y Wu, JL Shohet, MP Richards 2022. Investigation of the heme crevice of bovine and trout IV hemoglobin with plasma induced modification of biomolecules (PLIMB). Reciprocal Meat Conference. Des Moines, IA, USA from June 12 to June 15, 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Richards, MP, James G. Whalin, Yuting Wu, Surendranath P. Suman, Yifei Wang and J. Leon. Shohet. 2022. Myoglobin and hemoglobin: discoloration, lipid oxidation and solvent access to the heme pocket. Meat and Muscle Biology, Vol. 6(3) 14400 1-7. https://doi.org/10.22175/mmb.14400
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Wu H, Park SY, Richards MP. Effects of sodium chloride and sodium tripolyphosphate on the prooxidant properties of hemoglobin in washed turkey muscle system. Food Chem X. 2022 Oct 13;16:100480. doi: 10.1016/j.fochx.2022.100480. PMID: 36277871; PMCID: PMC9583034.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Mark P. Richards. Role of proteins in lipid oxidation Session. Aspects of myoglobin and hemoglobin-mediated lipid in muscle foods and its inhibition. American Oil Chemists Society (AOCS) Annual Meeting. Denver, CO. April 30-May 3, 2023.
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2023
Citation:
Ha Nguyen, Haoyu Cheng, Yuting Wu, Yoann Choy, Benjamin B Minkoff, Thao T. Nguyen, Mark P. Richards, Michael R. Sussman, Hau D. Le, and J. Leon Shohet. Gas-Bubble Mixing Improves Liquid Treatment with Cold Atmospheric Plasma. Plasma Science Conference 2023.
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Progress 04/01/21 to 03/31/22
Outputs
Target Audience:The target audience includes individuals working in the following fields: meat science, electrical engineering, biochemistry, mass spectroscopy, food science, protein crystallography, biophysical chemistry, seafood technology, aquaculture, and the meat industry. Specific organizations with interest include the American Meat Science Association (AMSA), Institute of Electrical and Electronics Engineers (IEEE), American Society for Mass Spectrometry (ASMS), Institute of Food Technologists (IFT), American Oil Chemists Society (AOCS), Poultry Science Association (PSA), Midwest Advanced Food Manufacturing Association (MAFMA), National Pork Producers Council (NPPC), North American Meat Institute (NAMI), National Cattlemen's Beef Association (NCBA), and the Trans-Atlantic Fisheries Association (TAFT). Knowledge from this project will be delivered via a graduate level Food Lipids course that is taught on the University of Wisconsin-Madison campus. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two graduate students are receiving training as part of this grant. This is a unique opportunity for cross-discipline work in fields encompassing agriculture, biochemistry, and electrical engineering. Advanced mass spectroscopy studies are part of this work, and a highly innovative software is being utilized to interpret the mass spec data. Lab group meetings occur headed by co-I Shohet that interface students of electrical engineering, mass spectroscopy and agricultural disciplines. How have the results been disseminated to communities of interest?The current findings are to be presented at the Reciprocal Meats Conference in June 2022. The findings will be presented in a poster and as an oral presentation to attendees. Previous findings were also reported at the RMC conference in August of 2021 in Reno, NV. Publications in peer-reviewed journals have also resulted from this project that are included in this report. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we will be making solvent access measurements at additional pH values to better understand how this environmental factor affects mechanisms of heme protein oxidation and heme loss. We will also evaluate a hydrogen peroxide scavenging approach that allow for longer times of plasma exposure without causing metMb formation. This will better allow measurement of solvent access to the heme pocket to investigate the mechanism of Mb oxidation. We will also explore the use of inert gas atmosphere during PLIMB which has the potential to better allow assessment of solvent access to the heme pocket. Some experiments will also be replicated to have more robust data sets and demonstrate the degree of reproducibility when using PLIMB.
Impacts
What was accomplished under these goals?
IMPACT: Food waste is a major agricultural and societal problem. Discoloration and off-flavor development during storage of muscle foods (e.g. meat, poultry, and fish) contribute to food waste. The focus of this project is to better understand the mechanisms that leads to discoloration and off-flavors in muscle foods. Through better understanding of the mechanisms involved, improved strategies to inhibit the quality deterioration can be realized. Our effort combines expertise in the fields of food biochemistry, electrical engineering, and advanced protein analytics to address the mechanisms involved. Solvent access to the heme pocket of the two color-imparting proteins of muscle has been proposed to incur discoloration and off-flavor formation, yet measurements of solvent access are lacking. The change in knowledge from our work as described below is that we have been successful in measuring attributes of solvent access to the heme pocket of these two proteins, namely myoglobin (Mb) and hemoglobin (Hb). This allows the role of solvent access to be better understood relative to other mechanisms of discoloration and off-flavor formation. From this effort, strategies to inhibit discoloration and off-flavor formation have become more focused. Our approach is to implement the use of plasma-induced modification of biomolecules (PLIMB). A dielectric barrier discharge generates the plasma and reactive species that chemically bond to solvent accessible sites of proteins. This approach now is not only implemented in biomedical fields (e.g. antibody research) but also in food quality research to counteract food waste. Determine solvent access to the distal and proximal heme crevices of bovine Mb and bovine Hb at pH 5.6 compared to pH 6.3. Low pH increases metMb formation yet the primary mechanism of metMb formation remains poorly understood. Bovine Mb was exposed to PLIMB for up to 3 seconds to assess solvent access to the heme pocket in relation to metMb formation. Circular dichroism measurements indicated that secondary structure was maintained during PLIMB which indicates the protein structure was minimally disturbed by the plasma exposure. The level of metMb formation during PLIMB was held to a low degree so to be able to assess the mechanism of metMb formation. Modifications to the heme pocket were determined using liquid chromatography tandem mass spectrometry and use of Byos Software (Protein Metrics). There was dose-dependent modification of His(E7) at the lower pH value but not the higher pH. His(E7) is the 7th residue along the E-helix and part of the heme pocket. This suggests the heme pocket was more exposed to solvent at the lower pH value. This experiment and some of the experiments below have been replicated and are currently being analyzed to have more robust data sets to interpret. Low pH increases metHb formation yet the primary mechanism of metHb formation remains poorly understood. Bovine Hb was exposed to PLIMB for up to 4 seconds. PLIMB did not disrupt protein secondary structure and metHb formation was kept low. There were dose-dependent modifications of αTyr(C7), αCE1 and αHis(F8) at both pH values. There were trends (p-values = 0.15 - 0.17) that the percent modification was higher at the lower pH value regarding αTyr(C7) and αHis(F8). These findings suggest the heme pocket was more exposed to solvent at the lower pH value. Determine solvent access to the distal heme cavity of bovine Mb compared to Mb containing covalently bound HNE. The binding of the lipid aldehyde 4-hydroxy-2-nonenal (HNE) is known to accelerate metMb formation. It may be that solvent access to the heme pocket is increased in Mb adducted with HNE to explain the increased metMb formation. After incubating Mb with HNE, approximately 75% of the Mb contained at least one molecule of bound HNE. Di-adducts and tri-adducts were also found to occur. Bovine Mb with and without HNE adduction was exposed to PLIMB for up to 0.75 seconds. Longer plasma exposures caused elevated metMb formation and so were avoided. Plasma exposure for up to 0.75 s did not disrupt protein secondary structure. There were no dose-dependent modifications to any sites of the heme pocket for either treatment. Dose-dependent modification did appear to take place at Phe(H15) but this was corrected to be modification at Met(H19) which is not part of the heme pocket. An approach to allow longer plasma exposure while limiting metMb formation has been developed and is to be implemented to better assess how solvent access to the heme pocket is affected by HNE adduction. Determine solvent access to the distal and proximal heme cavity of turkey HbA compared to turkey HbA containing covalently bound caffeic acid. This objective was not pursued in the current reporting year. Although we have obtained some valuable insight regarding solvent access to the heme pocket of bovine Mb, the limited time of plasma exposure before metMb formation occurs has limited our measurements of solvent access. We are currently planning additional studies with bovine Mb at conditions that allow for increased time of plasma exposure while limiting metMb formation. This further evaluation of bovine Mb seems a better use of resources than working towards obtaining a somewhat narrow evaluation of the turkey hemoglobin. Determine solvent access to the distal and proximal heme cavities of bovine Mb compared to bovine Hb and bovine Hb to trout IV Hb. We have made progress in examining the role of solvent access to the heme pocket of trout IV Hb as pH is varied. This fish Hb is mostly in the deoxyHb form at pH 6.5 while at pH 7.2 the protein is nearly all in the oxyHb form. Plasma exposures were for up to 2 seconds. Exposure beyond 2 s incurred elevated metHb content which would limit studying the effect of solvent access on Hb oxidation. There was dose-dependent modification of αPhe(G5) at both pH values. When comparing pH, there was more modification of αPhe(G5) at the higher pH value. This finding suggests deoxyHb decreases solvent access to the heme pocket relative to oxyHb, consistent with an outer sphere mechanism mediated by the reaction of deoxyHb with O2 to form metHb. Since Hb oxidation occurs faster at low pH, these findings suggest the outer sphere mechanism of Hb oxidation involving deoxyHb is more impactful than the inner sphere mechanism involving solvent access to the heme pockets of oxyHb. We have made progress examining the role of solvent access to the heme pocket of bovine metHb compared to trout IV metHb in relation to the rapid heme dissociation from the fish Hb. Our studies this year focused on comparing solvent access to the heme pocket of each metHb at pH 6.5. There are 17 residues within four angstroms of the heme moiety in Hb which represent the heme pocket. Of those, nine residues in bovine α-chain and three residues in bovine β-chain had validated modifications. Only one residue, αTyr(C7) was modified dose-dependently in bovine Hb. In trout IV methemoglobin, four residues were validated in α-chain and three in β-chain, with two residues being dose dependent, αTyr(C7) and αHis(CE3). When making the specie comparison, αTyr(C7) was more modified in bovine metHb whereas αHis(CE3) was more modified in the fish metHb. We are currently developing an explanation for the differential modification at αC7 and αCE3 by specie. The fact that solvent access to the heme pocket was not clearly higher in trout IV metHb suggests some mechanism other than solvent access to the heme pocket explains the low heme affinity in the fish Hb.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2021
Citation:
JG Whalin, PS Surendranath, Y Wang, JL Shohet, and MP Richards. Using PLIMB generated hydroxyl radicals to determine solvent access to the heme pocket of HNE adducted bovine myoglobin. Reciprocal Meat Conference to be held in Reno, Nevada, USA from August 15 to August 18, 2021.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2022
Citation:
Wu H, Yin, J, Xiao S, Yin J, Richards MP, Zhang J. (2022). Quercetin as an inhibitor of hemoglobin-mediated lipid oxidation: mechanisms of action and use of molecular docking. doi: 10.1016/j.foodchem.2022.132473
- Type:
Journal Articles
Status:
Accepted
Year Published:
2022
Citation:
Wu, H. Richards, MP, Undeland, I. Lipid oxidation and antioxidant delivery systems in muscle food. (2022). Comprehensive Reviews in Food Science and Food Safety. 125. https://doi.org/10.1111/1541-4337.12890
- Type:
Journal Articles
Status:
Accepted
Year Published:
2021
Citation:
Kassa T, Whalin JG, Richards MP, Alayash AI. Caffeic acid: an antioxidant with novel antisickling properties. FEBS Open Bio. 2021 Sep 12. https://doi:10.1002/2211-5463.13295
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Progress 04/01/20 to 03/31/21
Outputs
Target Audience:The target audience includes individuals working in the following fields: meat science, electrical engineering, biochemistry, mass spectroscopy, food science, protein crystallography, biophysical chemistry, seafood technology, aquaculture, and the meat industry. Specific organizations with interest include the American Meat Science Association (AMSA), Institute of Electrical and Electronics Engineers (IEEE), American Society for Mass Spectrometry (ASMS), Institute of Food Technologists (IFT), American Oil Chemists Society (AOCS), Poultry Science Association (PSA), Midwest Advanced Food Manufacturing Association (MAFMA), National Pork Producers Council (NPPC), North American Meat Institute (NAMI), National Cattlemen's Beef Association (NCBA), and the Trans-Atlantic Fisheries Association (TAFT). Knowledge from this project will be delivered via a graduate level Food Lipids course that is taught on the University of Wisconsin-Madison campus. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two graduate students are receiving training as part of this grant. This is a unique opportunity for cross-discipline work in fields encompassing agriculture, biochemistry, and electrical engineering. Advanced mass spectroscopy studies are part of this work and a highly innovative software is being utilized to interpret the mass spec data. Lab group meetings occur headed by co-I Shohet that interface students of electrical engineering, mass spectroscopy and agricultural disciplines. Due to the pandemic-related issues, one graduate student has worked on the project only for the first 3 months of this reporting period. How have the results been disseminated to communities of interest?The current findings are to be presented at the Reciprocal Meats Conference in August 2021 as a virtual conference. Findings from this project will be presented in a graduate course entitled Chemistry of Food Lipids taught at the University of Wisconsin-Madison. What do you plan to do during the next reporting period to accomplish the goals?Solvent access experiments comparing Mb to Mb-HNE will be repeated to further establish how adduction of HNE to Mb affects solvent access to the heme crevice in relation to metMb formation. Solvent access experiments comparing bovine Hb at pH 5.7 to 6.3 will be done in relation to metHb formation. Solvent access experiments comparing bovine Hb to trout IV Hb will be done in relation to metHb formation. Solvent access experiments comparing bovine metHb to trout IV metHb will be done in relation to heme dissociation.
Impacts
What was accomplished under these goals?
First, we made progress examining the role of solvent access to bovine myoglobin (Mb) compared to Mb adducted with 4-hydroxy-2-nonenal (HNE). We have determined that HNE binds at histidine residues of Mb including His24, His36, His88, His119 and His152. Mb and Mb-HNE were exposed to plasma to probe solvent access to the heme pocket. Three sites in the proximal cavity were modified compared to two in the distal cavity. The proximal Phe138, the 15th site in the H-helix, was the most modified. Statistical evaluations indicated a trend that solvent access was greater in Mb compared to Mb-HNE. This suggested adduction by HNE caused structural perturbations that increase metMb formation (e.g. discoloration) by a mechanism other than facilitating solvent entry into the distal heme crevice to facilitate proton-mediated metMb formation. This alternative mechanism is being currently explored. Second, we made progress in examining the role of pH on solvent access to the heme pocket of bovine hemoglobin (Hb). MetHb formation was much more rapid at pH 5.7 compared to pH 6.5 in a buffer that was validated to hold these pH values during PLasma-Induced Modification of Biomolecules (PLIMB). We have established a plasma exposure regime with acceptable limits of metHb formation during plasma exposure so that the degree of solvent access to bovine Hb at each pH value can be examined. Third, we made progress in examining the role of solvent access to the heme pocket of bovine Hb compared to trout IV Hb. The fish Hb oxidizes to metHb much more rapidly than the mammalian Hb, and it is suspected solvent access to the heme pocket may differ between the two heme proteins to explain the disparate rates of metHb formation. We have established a plasma exposure regime with acceptable limits of metHb formation during plasma exposure so that the degree of solvent access to both heme proteins can be examined. Initial mass spectrometry findings indicate a different protease cocktail will be needed to assess solvent access in trout IV Hb. Fourth, we made progress in examining the role of solvent access to the heme pocket of bovine metHb compared to trout IV metHb in relation to the rapid heme dissociation from the fish Hb. We have established a plasma exposure regime with acceptable limits on heme degradation and structural disruption to examine solvent access to both heme proteins.
Publications
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Progress 04/01/19 to 03/31/20
Outputs
Target Audience:The target audience includes individuals working in the following fields: meat science, electrical engineering, biochemistry, mass spectroscopy, food science, protein crystallography, biophysical chemistry, seafood technology, aquaculture, and the meat industry. Specific organizations with interest include the American Meat Science Association (AMSA), Institute of Electrical and Electronics Engineers (IEEE), American Society for Mass Spectrometry (ASMS), Institute of Food Technologists (IFT), American Oil Chemists Society (AOCS), Poultry Science Association (PSA), Midwest Advanced Food Manufacturing Association (MAFMA), National Pork Producers Council (NPPC), North American Meat Institute (NAMI), National Cattlemen's Beef Association (NCBA), and the Trans-Atlantic Fisheries Association (TAFT). Knowledge from this project will be delivered via a graduate level Food Lipids course that is taught on the University of Wisconsin-Madison campus. Changes/Problems:One graduate student is currently detained in their home country due to the pandemicso some delays may occur regarding objective 1 that is to be completed by that student. What opportunities for training and professional development has the project provided?Two graduate students are receiving training as part of this grant. This is a unique opportunity for cross-discipline work in fields encompassing agriculture, biochemistry, and electrical engineering. Advanced mass spectroscopy studies are part of this work and a highly innovative software is being utilized to interpret the mass spec data. Weekly lab group meetings occur headed by co-I Shohet that interface students of electrical engineering, mass spectroscopy and agricultural disciplines. Collaborator Sussman in Biochemistry also holds lab group meetings that are attended by graduate students, post docs, and technicians from the differing disciplines. How have the results been disseminated to communities of interest?The current findings are to be presented at the International Congress of Meat Science and Technology in August 2020 as a virtual conference. Another abstract was accepted for the ICPM 2020: International Conference on Plasma Medicine but conference was cancelled due to pandemic. Findings from this project will be presented in a graduate course entitled Chemistry of Food Lipids taught at the University of Wisconsin-Madison. What do you plan to do during the next reporting period to accomplish the goals?Bovine hemoglobin will be prepared to complete preparation of the proteins necessary for the project. Completion of objective 1 and 2 are anticipated of being completed. One student is currently detained in their home country due to the pandemicso some delays may occur regarding objective 1 that is to be completed by that student.
Impacts
What was accomplished under these goals?
We have established most of the methods that are required to carry out the objectives of the project. These methods include i) preparing bovine myoglobin, turkey hemoglobin A and trout Hb IV, ii) establishing buffer conditions during protein modification since pH comparisons are part ofObjective 1and pH must vary littleduring exposure of Mb to the plasma, iii) alkylation experiments that covalently bond hydroxynonenal (HNE) to bovine Mb, iv) tryptic digest step to prepare peptides for mass spec, v) running of mass spec, and vi) mass spec data analysis to make assignments of protein modifications due to plasma exposure so to measure solvent access to the heme pocket. Various times of plasma exposure to Mb were done to establish upper limits of exposure so that solvent access could be assessed without extensive loss of protein structure. Initial experiments were carried out to establish the coordination of the multiple analyses that included the plasma exposure followed by metMb determination, secondary structure assessment, tryptic digestion of Mb, and the mass spec run.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
James Whalin, Daniel Benjamin, Faraz A. Choudhury, Yuting Wu, Yifei Wang, Surendranath P. Suman, J. L. Shohet, and Mark P. Richards. Hydroxyl radical footprinting of bovine myoglobin using plasma induced modification of biomolecules (PLIMB). International Congress of Meat Science and Technology (Aug 2-6, 2020) Virtual conference.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Yuting Wu, Faraz Choudhury, Daniel Benjamin, James Whalin, Joshua M. Blatz, J. Leon Shohet, Michael R. Sussman and Mark Richards. Plasma-Induced Modification of Biomolecules: A New Tool for Analysis of Protein Structures" has been accepted for "ICPM 2020: International Conference on Plasma Medicine" to be held on Aug 10-11, 2020 in New York, USA (Cancelled due to Covid-19 pandemic).
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