Article pubs.acs.org/JAFC
Covalent Binding of 4‑Hydroxy-2-nonenal to Lactate Dehydrogenase Decreases NADH Formation and Metmyoglobin Reducing Activity Ranjith Ramanathan,† Richard A. Mancini,*,† Surendranath P. Suman,‡ and Carol M. Beach§ †
Department of Animal Science, University of Connecticut, 3636 Horsebarn Road Extension, Unit 4040, Storrs, Connecticut 06249-4040, United States ‡ Department of Animal and Food Sciences, University of Kentucky, Lexington, Kentucky 40546, United States § Proteomics Core Facility, University of Kentucky, Lexington, Kentucky 40506, United States ABSTRACT: Lactate dehydrogenase (LDH) activity can regenerate NADH, which is a critical component in metmyoglobin reduction. However, limited research has determined the effects of lipid oxidation products on LDH activity. The overall objective of this study was to determine the effects of 4-hydroxy-2-nonenal (HNE) on LDH activity. LDH was reacted with HNE at pH 5.6 and 7.4, and LDH activity was measured as NADH formation following the addition of lactate and NAD. The effects of HNE on NADH-dependent metmyoglobin reduction also were analyzed. Mass spectrometric examination revealed that HNE adducts to LDH at both pH 5.6 and 7.4. More specifically, HNE binds with cysteine and histidine residues of LDH at pH 5.6 and 7.4. Covalent binding of HNE decreased NADH formation and metmyoglobin reduction (P < 0.05). These results indicate that secondary lipid oxidation products can inactivate enzymes involved in metmyoglobin reduction and have the potential to increase beef discoloration. KEYWORDS: beef color, metmyoglobin reduction, lipid oxidation, LDH, NADH-dependent cytochrome b5 reductase, mass spectrometry
■
INTRODUCTION Myoglobin is a water-soluble sarcoplasmic protein that can exist in one of three redox forms; namely deoxy-, oxy-, or metmyoglobin. Predominant oxymyoglobin on the surface of meat provides a consumer-preferred bright cherry-red color. Although several factors can influence the oxidation of oxymyoglobin to metmyoglobin, meat has an inherent ability to delay discoloration via metmyoglobin reduction, which is associated with the addition of an electron to metmyoglobin via enzymatic or nonenzymatic processes.1,2 NADH is an important cofactor involved in metmyoglobin reduction and therefore regeneration of NADH is critical for extending the flavor and color stability of meat. 4-Hydroxy-2-nonenal (HNE) is an α,β unsaturated aldehyde that can be formed from the oxidation of n-6 linoleic acid and arachidonic acid. More specifically, HNE is formed from unsaturated fatty acids present in intramuscular fat and mitochondrial membranes. Free and bound HNE have been reported in beef and HNE concentration is often considered an indicator of muscle food quality.3,4 4-Hydroxy-2-nonenal formed from lipid oxidation is very stable and can diffuse into the sarcoplasm from the site of production. Furthermore, HNE is very reactive and can influence the function of enzymes such as glucose-6-phosphate dehydrogenase,5 pyruvate dehydrogenase,6 glutathione reductase,7 glutathione transferase,8 and cytochrome c oxidase.9 More specifically, the electrophilic C-3 within HNE can covalently bind via Michael addition to histidine, lysine, or cysteine.10 To date, no research has evaluated the effects of HNE on lactate dehydrogenase (LDH) activity. In post-mortem muscle, enzymes involved in the glycolytic and tricarboxylic cycle retain activity, and research suggests that © 2014 American Chemical Society
post-mortem muscle can regenerate NADH via LDH activity.11−14 As a result, the NADH formed from LDH activity can be used for both cytochrome b5 reductase- and electron transport-mediated metmyoglobin reduction.15 Although LDH can play a significant role in the production of reducing equivalents necessary for improved color stability, no research has assessed the effect of oxidative processes on LDH activity, NADH production, and metmyoglobin reduction. Recent research has shown that preincubation of HNE with bovine mitochondria can decrease oxygen consumption and electron transport-mediated metmyoglobin reduction.16 Hence, given the location of LDH in both the mitochondria and sarcoplasm, it is possible that HNE formed from lipid oxidation of membrane lipids can influence enzyme activity in vitro. Our hypothesis was that reactivity of HNE toward nucleophilic amino acids will decrease LDH activity. Hence, the objectives of this study were to determine the effects of HNE on LDH activity, metmyoglobin reducing activity, and covalent binding to LDH using mass spectrometry.
■
MATERIALS AND METHODS
Materials and Chemicals. LDH (L-lactic dehydrogenase from bovine heart, type III, ammonium sulfate suspension, ≥ 500 units/mg protein), sodium monophosphate, Sephacryl 200HR, ammonium bicarbonate, ammonium sulfate, Tris-HCl, EDTA, sodium citrate, ethanol, acetonitrile, NAD, sodium borohydride, formic acid, trypsin, and trifluoroacetic acid were obtained from Sigma Chemical Co. (St. Received: Revised: Accepted: Published: 2112
November 4, 2013 January 28, 2014 January 31, 2014 January 31, 2014 dx.doi.org/10.1021/jf404900y | J. Agric. Food Chem. 2014, 62, 2112−2117
Journal of Agricultural and Food Chemistry
Article
Figure 1. ESI-MS spectrum of bovine LDH control (0.2 mM) at pH 7.4. The X-axis indicates mass measured as atomic mass unit (amu) and Y-axis indicates intensity measured as counts per second (cps).
Figure 2. ESI-MS spectrum of bovine LDH (0.2 mM) incubated with (a) HNE 0.04 mM and (b) 0.4 mM at pH 7.4. The X-axis indicates mass measured as atomic mass unit (amu) and Y-axis indicates intensity measured as counts per second (cps). Louis, MO). 4-Hydroxy-2-nonenal was obtained from Cayman Chemical Co. (Ann Arbor, MI). PD-10 columns, CM and DEAE cellulose ion exchange chromatography columns were obtained from Pharmacia (Piscataway, NJ). All chemicals were of reagent grade or greater purity. Myoglobin Isolation and Preparation of Metmyoglobin. Beef myoglobin was purified via ammonium sulfate precipitation and gel filtration chromatography according to Faustman and Phillips.17 The isolated myoglobin was a mixture of oxy-, deoxy-, and metmyoglobin. To limit the interference with metmyoglobin reduction in the current study, oxidizing agents were not used to convert oxy- or deoxymyoglobin to metmyoglobin. Hence, metmyoglobin was prepared by incubating myoglobin at 37 °C for 24 h. Columns precalibrated with either 50 mM phosphate buffer (pH 7.4) or 50 mM
phosphate buffer (pH 5.6) were used to adjust the pH of metmyoglobin solutions. The initial metmyoglobin content was 98.9%, determined according to Tang et al.18 Isolation of NADH-Dependent Cytochrome b5 Reductase. Metmyoglobin reductase was isolated according to Hagler et al.19 and Faustman et al.20 Aliquots (1 mL) were stored at −80 °C until use. Effects of HNE on Lactate Dehydrogenase Activity. Commercially purchased purified bovine heart LDH (0.2 mM) was incubated with HNE (0, 0.04, or 0.4 mM dissolved in ethanol) for 5 h at pH 7.4 or for 72 h at pH 5.6 (normal meat pH) in a glass test tube. Lactate dehydrogenase activity was measured as the formation of NADH at 0, 1, 3, and 5 h at pH 7.4 and 0, 24, 48, and 72 h at pH 5.6. Briefly, lactate (50 mM), LDH (0.2 mM), and NAD (0.2 mM) were reacted in incubation buffer (50 mM phosphate buffer) at both pH 2113
dx.doi.org/10.1021/jf404900y | J. Agric. Food Chem. 2014, 62, 2112−2117
Journal of Agricultural and Food Chemistry
Article
Figure 3. ESI-MS spectrum of bovine LDH (0.2 mM) incubated with (a) HNE 0.04 mM and (b) 0.4 mM at pH 5.6. The X-axis indicates mass measured as atomic mass unit (amu) and Y-axis indicates intensity measured as counts per second (cps).
Table 1. Summary of MS/MS Analysis Used to Determine HNE Adduction to LDH at pH 5.6 and 7.4 pH
treatment (mM)
5.6
HNE 0.04 HNE 0.4
7.4
HNE 0.04 HNE 0.4
peptide
peptide sequence
HNE modification
precursor peptide m/z
trypsin trypsin trypsin
280−299 159−170 280−299
GMYGIENEVFLSLPCILNAR VIGSGCNLDSAR GMYGIENEVFLSLPCILNAR
C294 C164 C294
2395 1347 2395
trypsin trypsin trypsin trypsin/Asp-N trypsin/Asp-N
159−170 159−170 280−299 65−77 179−195
VIGSGCNLDSAR VIGSGCNLDSAR GMYGIENEVFLSLPCILNAR DLQHGSLFLQTPK LGIHPSSCHGWILGEHG
C164 C164 C294 H68 C186
1347 1347 2395 1640 1956
enzyme used for peptide digestion
ethanol described in the previous section). Metmyoglobin reduction (nanomoles of metmyoglobin reduced per minute per milligram of reductase) was calculated according to Faustman et al.20 using the change in absorbance at 580 nm. The protein concentration of reductase was determined using a bicinchoninic acid protein assay. Mass Spectrometry. Protein masses of native and HNE-treated LDH, including LDH peptide preparation and analysis (Trypsin digestion), were measured according to the method described by Nair et al.23 For Asp-N and double digestion, 10 μL of LDH solution was dried and digested in 10 μL of 40 mM ammonium bicarbonate +9% acetonitrile containing 0.02 μg (Sigma, St. Louis, MO) proteomics grade endoproteinase Asp-N at room temperature for 4 h. Statistical Analysis. The experimental design was a completely randomized design with repeated measures, and each experiment was replicated five times (n = 5). Fixed effects for both NADH-dependent
values in a quartz cuvette for 3 min. The formation of NADH was measured according to Bergmeyer21 and Ramanathan et al.,22 where an increase in absorbance at 340 nm indicates NADH formation. Following determination of LDH activity, a PD column was used to remove unbound HNE and LDH was stored at −80 °C until use. NADH-Dependent Cytochrome b5 Metmyoglobin Reduction. The methodology according to Reddy and Carpenter2 was modified to determine NADH-dependent cytochrome b5 metmyoglobin reduction. The assay mixture contained 0.15 mM metmyoglobin in either 50 mM phosphate buffer at pH 5.6 or 7.4, 0.05 mL of 0.2 mM NAD, 0.05 mL of 1 M lactate, 0.1 mL of 5 mM EDTA, 0.1 mL of 3.0 mM potassium ferrocyanide, and 0.03 mL of purified NADHdependent cytochrome b5 reductase. The reaction was initiated by the addition of LDH at 25 °C according to Ramanathan et al.15 (0.02 mM LDH in the final reaction mixture; LDH preincubation with HNE or 2114
dx.doi.org/10.1021/jf404900y | J. Agric. Food Chem. 2014, 62, 2112−2117
Journal of Agricultural and Food Chemistry
Article
cytochrome b5 reductase and LDH activity include HNE concentration, incubation time, and their interaction. Data was analyzed using the MIXED Procedure of SAS (version 9.1, SAS Institute Inc. Cary, NC), and the Repeated option was used for both NADH-dependent cytochrome b5 reductase and LDH activity measurements. The diff option (LSD at P < 0.05 for significance) was used to separate leastsquares means from protected F-tests (P < 0.05).
■
RESULTS AND DISCUSSION Effects of HNE on Covalent Binding on LDH. Characterization of LDH using MS revealed that the mass of
Table 2. Active Site Residues in Bovine Heart Lactate Dehydrogenase active site
position in the sequence
amino acid
NAD binding substrate binding NAD/substrate binding substrate binding proton acceptor substrate binding
100 106 139 170 194 249
arginine arginine asparagine arginine histidine threonine
protein samples used in the current study matched that of LDH B (isoform predominantly present in cardiac muscle; Figure 1). Mass spectrometric examination revealed that HNE adducted to LDH. Mono-, di-, and triadducts were detected at pH 7.4, whereas only monoadducts were identified at pH 5.6 (Figures 2 and 3). More specifically, MS/MS results indicate that HNE can bind with cysteine residues of LDH at pH 5.6 and 7.4 (Table 1). Carbon 3 within HNE can covalently bind via Michael addition to histidine residues within myoglobin from all livestock and poultry species and result in increased oxymyoglobin oxidation.24−27 In addition, HNE adduction makes myoglobin a poor substrate for enzymatic metmyoglobin reduction.28 Furthermore, covalent binding of HNE to myoglobin can increase heme release.29 The active site residues involved in LDH enzymatic activity are summarized in Table 2. Although HNE did not bind to residues within the active sites, the current study suggests that binding to other amino acids can influence enzyme activity. For example, the amino acid sequence of LDH has 7 histidine, 5 cysteine, and 26 lysine residues (Figure 4; www.expasy.org).
Figure 5. Effect of HNE on LDH activity at pH 5.6 and 7.4. Standard error bars are indicated.
The MS/MS analysis in the current research indicates that HNE binds to cysteine and histidine residues (Table 1). More specifically, at 0.4 mM HNE, four sites of HNE adduction were identified at His-68, Cys-164, Cys-186, and Cys-294 at pH 7.4. However, no histidine adducts were identified at pH 5.6, likely because histidine residues would be expected to have a greater positive charge at pH 5.6 than 7.4, which would reduce reactivity with HNE.30 The current study is the first to report
Figure 4. Primary structure of bovine LDH. 2115
dx.doi.org/10.1021/jf404900y | J. Agric. Food Chem. 2014, 62, 2112−2117
Journal of Agricultural and Food Chemistry
■
Article
AUTHOR INFORMATION
Corresponding Author
*Phone: +1 860 486 1775. Fax: +1 860 486 4375. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
Mass spectrometric analysis was performed at the University of Kentucky’s Proteomics Core Facility, supported in part by funds from the Office of the Vice President for Research.
■
ABBREVIATIONS USED LHD, L-lactic dehydrogenase; NAD, nicotinamide adenine dinucleotide; NADH, reduced form, nicotinamide adenine dinucleotide; HNE, 4- hydroxy-2-nonenal; MS, mass spectrometry; amu, atomic mass unit; cps, counts per second
■
REFERENCES
(1) Ledward, D. A. Post-slaughter influences on the formation of metmyoglobin in beef muscles. Meat Sci. 1985, 15, 149−171. (2) Reddy, I. M.; Carpenter, C. E. Determination of metmyoglobin reductase activity in bovine skeletal muscles. J. Food Sci. 1991, 56, 1161−1164. (3) Sakai, T.; Kuwazuru, S.; Yamauchi, K.; Uchida, K. A. Lipid oxidation-derived aldehyde, 4-hydroxy-2-nonenal and omega-6 fatty acid contents in meats. Bio. Biotechnol. Biochem. 1995, 59, 1379−1380. (4) Lynch, M. P.; Faustman, C.; Silbart, L. K.; Rood, D.; Furr, H. C. Detection of lipid-derived aldehydes and aldehyde:protein adducts in vitro and in beef. J. Food Sci. 2001, 66, 1093−1099. (5) Szweda, L. I.; Uchida, K.; Tsai, L.; Stadtman, E. R. Inactivation of glucose-6-phosphate dehydrogenase by 4-hydroxy-2-nonenal. J. Biol. Chem. 1993, 268, 3342−3347. (6) Patel, M. S.; Korotchkina, L. G. Pyruvate dehydrogenase complex as a marker of mitochondrial metabolism. Inhibition by 4-hydroxy-2nonenal. Methods Mol. Biol. 2002, 186, 255−263. (7) Jagt, D. L. V.; Hunsaker, L. A.; Jagt, T. J. V.; Gomeg, M. S.; Gonzales, D. M.; Deck, L. M. Inactivation of glutathione reductase by hydroxynonenal and other endogenous aldehydes. Biochem. Pharmacol. 1997, 53, 1133−1140. (8) Drake, J.; Petroze, R.; Castegna, A.; Ding, Q.; Keller, J. N.; Markesbery, W. R. 4-Hydroxynonenal oxidatively modifies histones: implications for Alzheimer’s disease. Neurosci. Lett. 2004, 356, 155− 158. (9) Chen, J.; Schenker, S.; Frosto, T. A.; Henderson, G. I. Inhibition of cytochrome c oxidase activity by 4-hydroxynonenal (HNE). Role of HNE adduct formation with the enzyme subunits. Biochim. Biophys. Acta 1998, 1380, 336−344. (10) Esterbauer, H.; Schaur, R. J.; Zollner, H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radical Biol. Med. 1991, 11, 81−128. (11) Watts, B. M.; Kendrick, J.; Zipser, M. W.; Hutchins, B.; Saleh, B. Enzymatic reducing pathways inmeat. J. Food Sci. 1966, 31, 855−862. (12) Saleh, B.; Watts, B. M. Substrates and intermediates in the enzymatic reduction of metmyoglobin in ground beef. J. Food Sci. 1968, 33, 353−358. (13) Stewart, M. R.; Hutchins, B. K.; Zipser, M. W.; Watts, B. M. Enzymatic reduction of metmyoglobin by ground beef. J. Food Sci. 1965, 30, 487−491. (14) Kim, Y. H.; Hunt, M. C.; Mancini, R. A.; Seyfert, M.; Loughin, T. M.; Kropf, D. H. Mechanism for lactate−color stabilization in injection-enhanced beef. J. Agric. Food Chem. 2006, 54, 7856−7862. (15) Ramanathan, R.; Mancini, R. A.; Naveena, B. M. Effects of lactate on bovine heart mitochondria-mediated metmyoglobin reduction. J. Agric. Food Chem. 2010, 58, 5724−5729.
Figure 6. Effect of HNE on LDH-mediated enzymatic metmyoglobin reduction at pH 5.6 and 7.4. Standard error bars are indicated.
that HNE can influence enzymes involved in metmyoglobin reduction. Previous studies have shown that HNE can covalently bind to enzymes and decrease activity. For example, Hussain et al.31 reported that HNE decreased rabbit muscle enolase activity in vitro. Covalent binding of HNE to cysteines, histidine, and lysine residues of glyceraldehyde 3-phosphate dehydrogenase and subsequent enzyme inactivation was reported by Uchida and Stadtman.32 In support, the current study indicates that HNE can bind to cysteine and histidine residues in LDH. Effects of HNE on LDH Activity and Metmyoglobin Reduction. There was a significant HNE × incubation time interaction for LDH activity (Figure 5). Decreased NADH formation in samples treated with HNE was supported by limited enzymatic metmyoglobin reduction (Figure 6). The effect of HNE on LDH activity was concentration dependent, and HNE had a greater effect on enzyme activity at pH 7.4 than at pH 5.6. Several studies have reported that lipid oxidation and myoglobin oxidation are interrelated. In addition to direct interaction between myoglobin and HNE, the current study indicates that lipid oxidation products decrease meat color stability by influencing enzymes involved in NADH production and metmyoglobin reduction. In conclusion, HNE can covalently bind to histidine and cysteine residues in LDH and will decrease NADH formation. The results indicate that secondary lipid oxidation products will inactivate enzymes involved in metmyoglobin reduction and, as a result, increase beef discoloration. 2116
dx.doi.org/10.1021/jf404900y | J. Agric. Food Chem. 2014, 62, 2112−2117
Journal of Agricultural and Food Chemistry
Article
(16) Ramanathan, R.; Mancini, R. A.; Suman, S. P.; Cantino, M. E. Effects of 4-hydroxy-2-nonenal on beef heart mitochondrial ultrastructure, oxygen consumption, and metmyoglobin reduction. Meat Sci. 2012, 90, 564−571. (17) Faustman, C.; Phillips, A. L. Measurement of discoloration in fresh meat. Current Protocols in Food Analytical Chemistry; Wiley: New York, 2001; Chapter F3, p F3.3. (18) Tang, J.; Faustman, C.; Hoagland, T. A. Krzywicki revisited: equations for spectrophotometric determination of myoglobin redox forms in aqueous meat extracts. J. Food Sci. 2004, 69, C717−C720. (19) Hagler, L.; Coppes, R. I., Jr.; Herman, R. H. Metmyoglobin reductase. Identification and purification of a reduced NADH dependent enzyme from bovine heart which reduces metmyoglobin. J. Biol. Chem. 1979, 254, 6505−6514. (20) Faustman, C.; Cassens, R. G.; Greaser, M. L. Reduction of metmyoglobinby extracts of bovine liver and cardiac muscle. J. Food Sci. 1988, 53, 1065−1067. (21) Bergmeyer, H. U. Methods of Enzymatic Analysis; Academic Press: New York, 1974; pp 545−546. (22) Ramanathan, R.; Mancini, R. A; Joseph, P.; Suman, S. P. Bovine mitochondrial oxygen consumption effects on oxymyoglobin in the presence of lactate as a substrate for respiration. Meat Sci. 2013, 93, 893−897. (23) Nair, M. N.; Suman, S. P.; Li, S.; Joseph, P.; Beach, C. M. Lipid oxidation-induced oxidation in emu and ostrich myoglobins. Meat Sci 2014, 96, 984−993. (24) Alderton, A. L.; Faustman, C.; Liebler, D. C.; Hill, D. W. Induction of redox instability of bovine myoglobin by adduction with 4-hydroxy-2-nonenal. Biochemistry 2003, 42, 4398−4405. (25) Suman, S. P.; Faustman, C.; Stamer, S. L.; Liebler, D. C. Proteomics of lipid oxidation-induced oxidation of porcine and bovine oxymyoglobins. Proteomics 2007, 7, 628−640. (26) Naveena, B. M.; Faustman, C.; Tatiyaborworntham, N.; Yin, S.; Ramanathan, R.; Mancini, R. A. Mass spectrometric characterization and redox instability of turkey and chicken myoglobins as induced by unsaturated aldehydes. J. Agric. Food Chem. 2010, 57, 8668−8676. (27) Yin, S.; Faustman, C.; Tatiyaborworntham, N.; Ramanathan, R.; Maheswarappa, N. B.; Mancini, R. A.; Joseph, P.; Suman, S. P.; Sun, Q. Species-specific myoglobin oxidation. J. Agric. Food Chem. 2011, 59, 12198−12203. (28) Lynch, M. P.; Faustman, C. Effect of aldehyde lipid oxidation products on myoglobin. J. Agric. Food Chem. 2000, 48, 600−604. (29) Faustman, C.; Richards, M. P.; Naveena, B. M.; Grunwald, E. M. Effect of HNE on bovine and porcine myoglobin redox stability and heme release. Institute of Food Technologists Annual Meeting, Anaheim, CA, June 6−9, 2009, . (30) Barrick, D.; Hughson, F. M.; Baldwin, R. L. Molecular mechanisms of acid denaturation. The role of histidine residues in the partial unfolding of apomyoglobin. J. Mol. Biol. 1994, 237, 588− 601. (31) Hussain, S. N. A.; Ghassan Matar, G.; Barreiro, E.; Florian, M.; Divangahi, M.; Theodoros Vassilakopoulos, T. Modifications of proteins by 4-hydroxy-2-nonenal in the ventilator muscles of rats. Am. J. Physiol.: Lung Cell Mol. Phys. 2006, 290, L996−L1003. (32) Uchida, K.; Stadtman, E. R. Covalent attachment of 4hydroxynonenal to glyceraldehyde-3-phosphate dehydrogenase. A possible involvement of intra- and intermolecular cross-linking reaction. J. Biol. Chem. 1993, 268, 6388−6393.
2117
dx.doi.org/10.1021/jf404900y | J. Agric. Food Chem. 2014, 62, 2112−2117
