1. Introduction
The pathways that initiate abnormal cell growth and transformation to a cancerous state require shifts in both metabolism and the microenvironment. In recent years, changes in cellular energy metabolism that drive cell growth and division have been investigated in greater detail [
1]. The role of lactate dehydrogenase (LDH) enzymes in this metabolic switch is especially important.
Early studies of LDH and lactate synthesis focused on its role in carbohydrate metabolism as an end product of anaerobic glycolysis. More recently, efforts have been directed at understanding the role of LDH enzymes in tumor growth [
2,
3]. LDH enzymes are overexpressed in many cancers, especially prior to and even when tumors receive adequate oxygen [
4]. More than 80 years ago, Otto Warburg observed enhanced glycolysis despite functionally intact mitochondria [
5,
6].
The two predominant LDH enzymes are cytosolic and were originally designated as muscle (LDH-A, the M form) or heart (LDH-B, the H form). Functional LDH is tetrameric and the two LDH enzymes can form complexes of M4, H4 or heterotetramers of both M and H [
7]. While LDH-A preferentially catalyzes the formation of L-lactate from pyruvate with the concomitant oxidation of NADH to NAD
+, LDH-B favors the reverse reaction. Their cellular expression even within tissues varies widely, with LDH-B expressed in neurons and LDH-A expressed in astrocytes [
8,
9]. Likewise, cancer-associated fibroblasts expressing LDH-A produce lactate and adjacent cancer cells expressing LDH-B consume lactate for energy in a symbiotic manner [
10]. Zha et al. reported increased LDH-B expression as a result of hyperactive mTOR signaling [
11]. Recently, Vlasiou et al. described their work on small molecule inhibitors of LDH-B as a strategy to treat breast and lung cancers [
12].
LDH enzymes have other functions in addition to their metabolic roles. A dimeric complex between LDH-A and the glycolytic enzyme GAPDH acts as a nuclear transcription factor [
13]. Also, LDH-A serves as a single-stranded DNA binding protein in an NADH-dependent manner [
14,
15]. A mitochondrial LDH isozyme has been identified in some prostate and liver cancer cells [
16].
L-lactate has been recognized as a signaling molecule with roles in immune cell function, histone modification and gene expression [
10,
17]. Lactate decreases immune cell function so that tumor cells evade detection. Millimolar concentrations of lactate inhibit histone deacetylases, thereby coupling a cell’s metabolic state with gene transcription [
18]. Lactate concentrations are typically 1.5 to 3 mM in blood and normal tissues but can rise to 20–30 mM in cancerous tissue [
17,
19]. Further, Ross et al. reported that levels of lactate in the brain increased in a mouse model of aging [
20]. As the ratio of LDH-B to LDH-A expression decreased, lactate increased. They suggest that increasing lactate concentrations in the brain may serve as a marker of age-related decline.
Beyond its pivotal role in cancer cell metabolism, LDH-A was identified as a target of oxidative stress in a neuronal cell line [
21]. Extensive pathologic and cellular data link the accumulation of protein oxidation to the development of neurodegenerative diseases [
22]. For example, myeloperoxidase (MPO), the enzyme that generates the strong oxidant hypochlorous acid (HOCl), is aberrantly expressed in neurons in Alzheimer’s disease brains [
23]. As a result, an MPO-specific oxidation product, 3-chlorotyrosine, increased threefold in Alzheimer’s brains relative to control brains.
The susceptibility of LDH enzymes to oxidation and inhibition by reactive oxygen species (ROS) is an important area of research. We recently reported that singlet oxygen (
1O
2) induced the formation of multiple crosslinks between LDH-A subunits, thereby inhibiting its activity [
24]. We were particularly interested in
1O
2 because it is the primary oxidant produced during photodynamic therapy [
25,
26]. We also investigated the effects of
1O
2 and other ROS on the cysteines of LDH-A [
27]. Cysteine labeling has been employed extensively in our research because it is often indicative of protein conformational changes.
To our knowledge, the effect of oxidants on LDH-B structure and activity has not been investigated. Given its critical role in both cancer cell metabolism and in the neuron-astrocyte lactate shuttle, we examined the effects of several ROS including
1O
2, HOCl, and H
2O
2 on LDH-B. Our interest focused on the cysteines of LDH-B because thiol oxidation is a likely outcome of cellular oxidative stress and is associated with degenerative disease progression [
28,
29]. Each of the four subunits of the LDH-B homotetramer has five reduced cysteines; one of these (cys164) is in close proximity to the nicotinamide of NAD(H) while another is adjacent to the active site [
30,
31].
Using SDS-PAGE in conjunction with a cysteine-specific fluorescent tag, kinetic assays, and native gel electrophoresis, we show that LDH-B is inhibited by
1O
2. Its susceptibility to oxidation and inhibition by
1O
2 is comparable to that of LDH-A. While HOCl did oxidize LDH-B cysteines and inhibited activity, higher HOCl concentrations were required relative to LDH-A. Like LDH-A, once LDH-B was oxidized by HOCl, the loss of activity was not reversed by disulfide reduction; therefore, damage to LDH-B by HOCl is irreversible. LDH-B was resistant to inhibition and to cysteine oxidation by H
2O
2, chloramines and Angeli’s salt, a cysteine-reactive nitroxyl donor [
32].
Lastly, the ability of LDH-B to enhance NADH oxidation, independent of pyruvate, was examined. Several research groups have reported that mammalian LDH-A enhances NADH oxidation following ROS exposure in vitro [
33,
34]. They determined that increased NADH oxidation by LDH-A involved a chain reaction propagated by superoxide anion (O
2•−) that yields H
2O
2. Wu et al. demonstrated in HeLa cells that both LDH-A and LDH-B were responsible for increased H
2O
2 production and overall oxidative stress [
35]. Herein, we show that
1O
2 initiated NADH oxidation by LDH-B was comparable to that of LDH-A.
2. Materials and Methods
Materials and Reagents. Maleimide-5-fluorescein (M5F) was from ThermoFisher, Waltham, MA, USA. Chlorin e6 and pheophorbide A (pheoA) were from Frontier Scientific, Logan, UT, USA. Solutions of chlorin e6 and pheoA were prepared in DMF and stored at −20 °C. Porcine heart LDH-B and rabbit muscle LDH-A were from EMD Millipore. All other chemicals were from Fisher or Sigma and were of the highest purity available. The concentration of HOCl was determined by measuring the absorbance at 292 nm (ε
292 = 350 M
−1 cm
−1) in 0.1 M NaOH [
36]. A solution of Angeli’s salt was prepared immediately prior to use in 0.01 M NaOH. Glycine chloramine was synthesized as described [
37]. All reactions were performed at 20–22 °C unless otherwise stated. To calculate LDH-A concentration, the absorbance at 280 nm is converted to mg/mL using 1.13 as a conversion factor (Worthington protein manual). For LDH-B, the absorbance at 280 nm was converted to molar using 55,040 M
−1 cm
−1 [
38].
LDH-B treatment with DTT and desalting. Lyophilized LDH-B (20–24 mg solid) was dissolved in 3 mL 10 mM phosphate buffer (PB) pH 7.4 containing 10 mM dithiothreitol (DTT). After 15 min, the LDH-B solution was desalted on a Bio-Rad 10DG column to remove DTT and any small molecule contaminants.
Photo-oxidation of LDH-B. LDH-B (10 μM) was combined with photosensitizers in 10 mM PB 7.4 under ambient oxygen conditions (20 μL rxn). A 36-watt red light composed of eighteen 2-watt LEDs was used for photochemical experiments. The wavelength of emitted light was 660 nm. The intensity of the red light was quantitated in lux and the light intensity as a function of distance from the light source to the samples was measured. All buffers were equilibrated to 20–22 °C to ensure no differences in dissolved O2.
Enhanced NADH oxidation by LDH-B; initiation by 1O2. Reactions (100 μL) contained 2.5 μM methylene blue (MB) and 200 μM NADH in 50 mM PB pH 7.1 in a 96-well plate. 1O2 formation was initiated with 10 s of red light. Additions included control and oxidant-treated LDH-A or LDH-B (10 μM) and superoxide dismutase (SOD) (10 U/100 μL). Absorbance at 340 nm was measured prior to and immediately after light exposure. Absorbance was measured every 5 min for 40 min then at 10 min intervals until 60 min total.
LDH-B activity assay. LDH-B (10 μM) in 10 mM PB pH 7.4 was treated with 25, 50, 100, and 150 μM HOCl for up to 30 min at 30 °C. Residual HOCl was scavenged with 0.2 mM S-methyl-cysteine prior to analysis. HOCl dilutions were prepared in water immediately prior to use and stored on ice. After HOCl treatment, samples were diluted 1:10 in 20 mM Tris pH 8.8. Assays (200 μL) contained 20 mM Tris pH 8.8, 25 nM LDH-B, 2 mM NAD+, and 4 mM lithium lactate. NADH oxidation was monitored at 340 nm in a 96-well plate for 4 min at 30 °C. Rates were determined from the linear portion of the activity curve. Slopes were converted to M/min using 3500 as the conversion factor (for the plate assay). NADH (ε = 6220 M−1 cm−1 at 340 nm) samples were scanned in a microcuvette and in the 96-well plate to determine the conversion factor (ε × path length)
Labeling of LDH-B cysteines with M5F. LDH-B (10 µM with a maximum of 50 µM cys) in 10 mM PB pH 7.4 was either irradiated or treated with HOCl. (reaction volume = 20 µL). For HOCl experiments, S-methyl-cys (0.2 mM) was added to scavenge unreacted HOCl. M5F (10 mM stock in DMF) was added to achieve a 10-fold molar excess relative to LDH-B cys (500 μM final) and samples were incubated at 37 °C for 30 min. Proteins were resolved by SDS-PAGE on 10% gels and fluorescent images were captured using a Bio-Rad Chemi-doc XRS imaging system.
Alternatively, M5F-labeled LDH-B (60 μL reactions) was precipitated with 80% ethanol, incubated on ice for at least 30 min and the protein pellet was collected at 16,000× g for 10 min. Pellets were washed twice with 80% ethanol and resuspended in 6 M guanidine HCl in 0.1 M Tris pH 8.8. Fluorescein in each protein sample was quantitated at 495 nm relative to a fluorescein standard curve also prepared in 6 M guanidine HCl in 0.1 M Tris pH 8.8.
Native gel electrophoresis of LDH-B with activity stain. LDH-B samples were treated with photosensitizers, oxidant scavengers, and HOCl as described above. Native gel electrophoresis was performed using 0.8% agarose gels. 20 mM Tris, 20 mM glycine, and 2 mM EDTA pH 9.5 was used to prepare the gels and as the running buffer. Sample loading buffer (6×) contained 25% glycerol in Tris/glycine/EDTA buffer. Gels were run for 45–60 min at 90 V. The LDH activity stain (20 mL) contained 0.75 mM NAD+, 25 mM lithium lactate, 8–10 mg nitroblue tetrazolium (NBT), and 1–2 mg phenazine methosulfate (PMS) in 100 mM Tris pH 8.6. Gels were incubated with activity stain solution for 5–10 min in the dark until activity bands were observed. The reaction was halted by washing the gel with 5% acetic acid solution.
3. Results
We reported that singlet oxygen induced the formation of higher MW protein crosslinks in mammalian LDH-A that were detected by SDS-PAGE under reducing conditions [
27]. Both the extent of crosslink formation and the decrease in LDH-A enzymatic activity were dependent on the
1O
2 concentration.
3.1. Photo-Oxidation of LDH-B
To test the susceptibility of porcine LDH-B to oxidation by
1O
2, the same combination of photosensitizer (MB), red light, and ambient oxygen was employed as for our LDH-A studies. As irradiation time increased, the extent of higher MW crosslinks in LDH-B increased (
Figure 1A). Based on comparison with the mobility of the MW standards, the predominant crosslinks of the 36 kDa LDH-B protein are dimers and trimers. Of note, the 36 kDa monomer runs below the 37 kDa standard; therefore, it is expected that an LDH-B dimer would run below the 75 kDa standard (
Figure 1A) and a trimer would run at or near 100 kDa. Only very faint crosslinks above 100 kDa are discernible in the 90 s exposure lane.
Figure 1B shows the corresponding LDH-B activity data for identical samples and irradiation times. As time increased, LDH-B activity decreased. LDH-B samples containing 20% D
2O were also assayed for activity because D
2O increases the reactivity of
1O
2 by extending its lifetime. This is attributed to the lower-frequency vibrational stretching mode of D
2O relative to H
2O [
39]. At all times tested, the inclusion of 20% D
2O increased the extent of LDH-B inhibition (
Figure 1B). D
2O would not be expected to alter the lifetimes of other ROS.
Native gel electrophoresis with activity stain detection was performed to assess any changes in LDH-B native structure and activity simultaneously.
Figure 1C shows that the
1O
2 oxidation of LDH-B did not alter the protein’s mobility and also shows a time-dependent decrease in LDH-B activity. To test the effects of longer red-light exposure, lane 4 in
Figure 1C shows only minimal LDH-B activity after 120 s (vs. 90 s in
Figure 1A,B).
The activity results shown in
Figure 1B,C differ slightly though both use NAD
+ and lactate as LDH-B substrates. Under the conditions employed for
Figure 1B, absorbance at 340 nm (NAD
+ reduction to NADH) was measured to determine an initial linear rate of reduction. For the activity stain used in
Figure 1C, NADH reacted with phenazine methosulfate (PMS) and nitroblue tetrazolium (NBT) to yield a reduced formazan that precipitated within the gel. While both assays showed light-dependent decreases in LDH-B activity, the time courses in
Figure 1B,C were not identical.
Because the crosslinks in
Figure 1A were detected under reducing conditions, they are not disulfides. Further, cysteine is not the preferred target for
1O
2 [
40]. Nonetheless, we were interested in changes to LDH-B cysteine availability as a result of
1O
2 damage because it can be indicative of a conformational change. We observed modestly increased LDH-A cysteine exposure in our prior work that was consistent with reports by Pamp et al. in their copper-mediated LDH-A oxidation work [
27,
41].
3.2. Cysteine Labeling of 1O2-Damaged LDH-B
The status of the LDH-B cysteines, five per monomer, following irradiation was assessed by reaction with a fluorescein-tagged maleimide, M5F. LDH-B was treated with MB and red light under ambient oxygen for up to 120 s as described for
Figure 1. Subsequent analysis of the fluorescein labeled proteins by SDS-PAGE was performed under reducing conditions. After imaging the fluorescent bands, the gel was stained with Coomassie blue.
Figure 2 shows that the M5F labeling of control (dark) LDH-B had no effect on its mobility. For samples exposed to red light, cysteine labeling of the 36 kDa monomer was observed as well as labeling of the higher MW crosslinks. This was expected because cysteines in the crosslinks were not oxidized by
1O
2. As the dose of red light and, consequently,
1O
2 increased, the intensity of the 36 kDa monomer bands decreased (observed in both the fluorescent and the Coomassie-stained images) but labeling of the crosslinks did not.
Fluorescein labeling of the higher MW bands was diffuse, making it difficult to determine total cysteine labeling by densitometry. Therefore, identical samples were prepared, and fluorescein-labeled LDH-B was precipitated with ethanol, dissolved in guanidine HCl, and the concentration of incorporated fluorescein was determined from its absorbance at 495 nm relative to a fluorescein standard curve. For dark controls, 2.1 ± 0.1 mol cys/mol LDH-B was detected. For LDH-B samples exposed to red light for up to 120 s, the total cys labeling remained unchanged (2.1 ± 0.2 mol cys/mol LDH-B).
Of note, in the Coomassie-stained gel in
Figure 2, there is greater staining of higher MW crosslinks above the dimer and trimer bands than was observed in
Figure 1A. This may be a result of better Coomassie staining or labeling with fluorescein may enhance stain binding to LDH-B. Regardless,
1O
2 oxidation of LDH-B resulted in laddering when individual monomers within the tetramer were crosslinked.
3.3. 1O2 Generation with Chlorophyll Metabolites
In addition to MB, other photosensitizers were tested for their effects on LDH-B. In particular, we are interested in the chlorophyll metabolites, pheophorbide A (pheoA) and chlorin e6. PheoA was employed in our prior work on LDH-A and similar molecules are used in photodynamic therapy to generate ROS [
24,
25].
PheoA and chlorin e6 were compared with MB for their ability to inhibit LDH-B and to induce the formation of protein crosslinks as a result of
1O
2 damage. Higher concentrations of both were required relative to MB to yield comparable extents of LDH-B inhibition (
Supplemental Figure S1A). We were particularly interested in possible band shifts in the native gel assay because both pheoA and e6 are hydrophobic, unlike MB, and we had evidence for some nonspecific protein association by pheoA in our LDH-A work [
24].
Regardless of the photosensitizer employed, there was no change in LDH-B mobility on the native gel. (
Supplemental Figure S1A). In all cases, LDH-B activity was inhibited as evidenced by reduced activity staining. Likewise, all three photosensitizers, coupled with red light and ambient oxygen, induced formation of the same LDH-B protein crosslinks on SDS-PAGE (
Supplemental Figure S1B).
3.4. LDH-B Protection by Ascorbate and Hydrocaffeic Acid (HCA)
To aid in estimating a rate constant for the reaction of LDH-B with
1O
2, we performed competition studies using well-characterized
1O
2 scavengers. This approach has been employed by us and others when direct rate constant determination is challenging. Ascorbate was chosen because it is very water soluble and has biological relevance [
42]. Ascorbate (250 and 500 μM) was mixed with LDH-B (10 μM) and MB prior to irradiation.
Figure 3A shows that the presence of ascorbate (lanes 3 and 4) decreased
1O
2 damage relative to the light sample (lane 2) because the intensity of the higher MW bands decreased. The intensity of the higher MW crosslinks when 500 μM ascorbate was included is equal to that of the control in lane 1. Ascorbate had no effect on control (dark) LDH-B.
When identical samples were analyzed by native gel with activity stain, both the concentrations of ascorbate tested afforded full protection and the activity stain was equivalent to the dark control (
Figure 3B). However, when pheoA was used as the photosensitizer, those ascorbate concentrations did not protect to the same extent, as evidenced by the lighter activity bands in lanes 7 and 8 of
Figure 3B. We attributed this to the association of pheoA with LDH-B, so that the
1O
2 was generated in closer proximity to the amino acid targets vs. the ascorbate scavenger that would be dissolved in the surrounding milieu.
Our prior work with LDH-A also showed variability in protection from
1O
2 by the catechol, hydrocaffeic acid (HCA), when MB and pheoA were directly compared. The varying ascorbate effect in
Figure 3B with MB and pheoA prompted us to also evaluate HCA as a
1O
2 scavenger. Further, we included chlorin e6 because it is also more hydrophobic than MB but more soluble than pheoA due to three carboxylates.
Figure 3C summarizes LDH-B protection from light-induced damage by 0.5 mM ascorbate or 1.5 mM HCA. At these concentrations and with MB as the photosensitizer, we observed the complete protection of LDH-B activity. When chlorin e6 or pheoA were used as photosensitizers, 0.5 mM ascorbate protected, but not to the same extent as for MB (green bars in
Figure 3C). The ascorbate result with pheoA is consistent with the native gel data in
Figure 3B. For chlorin e6 and pheoA, 1.5 mM HCA afforded some protection, but it was not statistically significant (gray bars in
Figure 3C). When we included 2 mM HCA, the highest concentration tested, in assays with chlorin e6 or pheoA, LDH-B activity was greater than with 1.5 mM HCA, but it was still not protected completely. Lastly, samples with pheoA and chlorin e6 contained 10% DMF, which had no effect on LDH-B activity.
3.5. LDH-B Competition Studies
Competition studies with additional
1O
2 scavengers were performed using MB as the photosensitizer. In addition to ascorbate and HCA, we tested glutathione (GSH) and EDTA. EDTA is a less well-characterized
1O
2 scavenger, but our recent work showed that it reacts with
1O
2 and produces H
2O
2 [
43,
44]. EDTA is a common buffer component; therefore, it is imperative to assess its ability to scavenge
1O
2.
Table 1 summarizes competition studies in which scavengers were mixed with LDH-B and MB prior to irradiation. It is clear that much lower concentrations of ascorbate and HCA afforded LDH-B protection vs. when chlorin e6 or pheoA were used as photosensitizers (
Figure 3C). Only 50 μM ascorbate effectively competed with 10 μM LDH-B for
1O
2 when MB was used as the photosensitizer. Likewise, 500 μM HCA afforded full protection, whereas at 1.5 mM, little to no protection was observed in
Figure 3C with chlorin e6 or pheoA.
EDTA (1 mM) also competed with 10 μM LDH-B for
1O
2. EDTA reacts with
1O
2 to generate the EDTA radical cation and superoxide anion with a rate constant of ~10
5 M
−1 s
−1 [
43,
44,
48]. Because the rate constant is smaller, a higher concentration of EDTA was required for protection. As expected, GSH was an effective
1O
2 scavenger comparable to HCA, though less reactive than ascorbate (
Table 1).
We also tested sodium azide (NaN
3) for its ability to protect LDH-B from
1O
2 because it is a well-established and specific
1O
2 scavenger [
47,
49].
Supplemental Figure S3 shows that 0.1 mM to 1 mM NaN
3 protected LDH-B activity as assessed by native gel with activity stain. Protection by NaN
3 was comparable to that of GSH (
Table 1).
Because we are interested in both LDH enzymes, we combined LDH-A and LDH-B and assessed the
1O
2 damage to each on a native gel. The LDH enzymes have very different isoelectric points; for pig heart LDH-B, it is 5.6 and for rabbit muscle LDH-A, it is 8.4–8.6. On a native gel, they are separable; however, under conditions used to optimize LDH-B migration, LDH-A remains in the sample wells. Nonetheless, we observed a loss of activity to both after irradiation (
Supplemental Figure S4). The presence of LDH-A did not protect LDH-B from loss of activity.
We examined the effects of other ROS on LDH-B. In particular, we were interested in cysteine thiol oxidation to disulfides because it is a common form of protein damage that is often repaired by reductase enzymes like thioredoxin reductase [
50]. Hydrogen peroxide (H
2O
2) did not inhibit LDH-B activity at concentrations up to 5 mM, nor did it induce formation of disulfides. This resistance to H
2O
2 oxidation was also observed for LDH-A [
27]. Likewise, several other oxidants including chloramines and Angeli’s salt had no effect on LDH-B activity.
3.6. HOCl Oxidation of LDH-B
LDH-B in PB was treated with increasing concentrations of HOCl and analyzed by native gel electrophoresis and by SDS-PAGE under reducing and nonreducing conditions. Amine-based buffers like Tris react with HOCl and must be avoided. The pKa of HOCl is 7.5; therefore, a mixture of HOCl and OCl− is present at pH 7.4. For consistency and simplicity, we use HOCl herein to represent both species.
We observed the dose-dependent HOCl inhibition of LDH-B by native gel electrophoresis (
Figure 4A). The range of concentrations corresponds to 5–15 equivalents of HOCl relative to the LDH-B concentration of 10 μM. Given that each subunit contains five cysteines, the total concentration of cysteine is 50 μM per subunit; however, not all cysteines are expected to be accessible to oxidants. Further, the amino acids methionine and tyrosine are likely targets for HOCl [
36,
51].
Figure 4B shows that higher MW crosslinks were formed by HOCl treatment. On the +βME side of the gel, there is a faint dimer band in the lanes treated with HOCl. This non-disulfide product is detectable in the control lane as well, though lighter than for the HOCl treated lanes. This is not unusual for commercially available, lyophilized proteins that may become oxidized during isolation. No additional non-disulfide crosslinks were generated by HOCl. The −βME side of the gel in
Figure 4B shows more intense staining of protein crosslinks relative to the +βME side. There is more smearing and a very high MW band at the top of the separating gel. Also, in the absence of βME, just below the monomer band for all samples, there is a second, lighter protein band. This is not a proteolytic oxidation product because it is also detected in the untreated control lane. Also, on the −βME side, the monomer band decreased in intensity as the dose of HOCl increased.
Figure 4C shows that the range of HOCl concentrations used to induce disulfide formation also resulted in a dose-dependent loss of LDH-B activity consistent with the native gel in
Figure 4A. The lactate-dependent reduction in NADH by the control and HOCl-treated LDH-B was measured at 340 nm. Only 20–30% of control activity remained after treatment with 150 μM HOCl. HOCl-induced inhibition could not be overcome with higher concentrations of either substrate, and no changes in the extent of inhibition were observed.
3.7. HOCl Oxidation of LDH-B Cysteines
As for 1O2, the status of the five LDH-B cysteines after HOCl oxidation was assessed by M5F labeling. To ensure that no HOCl remained, it was scavenged with S-methyl-cysteine, a more soluble methionine analog, prior to M5F addition. The extent of M5F incorporation was assessed by SDS-PAGE of fluorescein-labeled LDH-B and by LDH-B precipitation with ethanol and quantitation of fluorescein from its absorbance at 495 nm vs. a fluorescein standard curve.
Figure 5 shows both the SDS-PAGE and quantitation results. LDH-B was treated with three concentrations of HOCl for either 15 or 30 min prior to M5F labeling. We observed both time- and dose-dependent decreases in M5F labeling of LDH-B in
Figure 5A. The time-dependent decreases in LDH-B cysteines treated with 100 or 150 μM HOCl did not change LDH-B activity. Cysteine oxidation continued but that did not further decrease LDH-B activity (15 vs. 30 min).
No time-dependent decrease in LDH-B cysteines was observed with 50 μM HOCl. This lower concentration of HOCl reacted completely with LDH-B in the first 15 min. Yellow thionitrobenzoic acid (TNB) reacts with HOCl and becomes colorless (oxidation to DTNB). When TNB was added to LDH-B samples treated with 50 μM HOCl for 15 min, the yellow color persisted, thereby confirming that none of the HOCl remained [
52].
The fluorescent monomer band decreased indicative of cysteine oxidation. No M5F labeling of higher MW bands was observed which is noteworthy. In this experiment, those cysteines of higher MW crosslinks are part of disulfides and, therefore, cannot be labeled with M5F. This is in contrast to the M5F labeling of higher MW bands that we observed for LDH-B oxidation by
1O
2 (
Figure 2). The crosslinks are observed in the Coomassie-stained gel (
Figure 5—right side) with the expected dimer and trimer bands and the general streaking as HOCl concentration increased. Also of note, the light bands detectable below the monomer in the stained gel were not modified by M5F even for the control, untreated LDH-B. There were no time- or oxidation-dependent shifts in the mobility of LDH-B, which is in contrast to our LDH-A results.
The quantitation of M5F labeling in
Figure 5B confirmed that HOCl oxidized LDH-B cysteines. The loss of LDH-B cysteines was dependent on the HOCl concentration. The control corresponds to 2.1 ± 0.1 mol cys/mol LDH-B, and at 150 μM HOCl, labeling decreased to 0.74 ± 0.04 mol cys/mol LDH-B.
Of the five cysteines per LDH-B monomer, two are located near the NAD
+/NADH binding domain. It was expected that the oxidation of these cysteines would inhibit LDH-B activity. The incubation of LDH-B with well-characterized thiol-reactive molecules inhibits LDH-B activity [
30]. We confirmed this using N-ethyl maleimide and iodoacetamide. The LDH-B modification of control LDH-B by M5F, a maleimide, also inhibited LDH-B. Lastly, the preincubation of LDH-B with NADH blocked M5F labeling. This shows that the cysteines protected by NADH are accessible to M5F.
Given that LDH-B cysteine oxidation to disulfides is an outcome of HOCl oxidation, we added a disulfide-reducing agent to try to restore LDH-B activity. When millimolar concentrations of DTT were added for up to 15 min after HOCl oxidation, we did not observe any reversal of the HOCl-induced inhibition of LDH-B. When Tris(2-carboxyethyl)phosphine (TCEP) was used instead of DTT, only a very modest 5–8% of activity was restored at each HOCl concentration.
Previously, we examined a unique reaction of LDH enzymes in which NADH oxidation is activated via a free radical mechanism that involves superoxide anion (or other ROS), is independent of pyruvate, and produces H2O2. We showed that 1O2 could activate enhanced NADH oxidation by rabbit muscle LDH-A and it was inhibited by superoxide dismutase. Once LDH-A was inhibited by 1O2 or HOCl oxidation, its ability to enhance NADH oxidation via this pathway was compromised. This was further evidence that 1O2 and HOCl oxidation affected the NADH binding event.
3.8. Enhanced NADH Oxidation by LDH-A and LDH-B
This reaction is of interest because the overexpression of LDH enzymes in tumors may lead to NADH depletion and ROS production via this mechanism. Therefore, we tested LDH-B for its ability to enhance NADH oxidation in this manner. The combination of MB, ambient oxygen, and red light (10 s) initiated NADH oxidation in the absence and presence of LDH-B. Brief light exposure yielded 3–4 μM 1O2 based on the resulting decrease in NADH absorbance at 340 nm. Once activated, NADH oxidation was monitored for 60 min.
Figure 6 shows the results of this experiment in which rabbit muscle LDH-A and pig heart LDH-B are directly compared. The essential role of MB is evident because NADH alone was stable and no NADH oxidation was observed. For MB alone (no LDH—red line), oxidation occurred at a linear rate and decreased to 146 μM NADH after 60 min (~1 μM /min).
Both LDH-A and LDH-B enhanced the rate of NADH oxidation relative to MB alone (
Figure 6). LDH-A was superior to LDH-B and only 29 μM NADH (of 200 μM) remained after 60 min, whereas for LDH-B, at 60 min, 55 μM NADH remained. The NADH oxidation curves for the two LDH enzymes are roughly parallel but the lag period for LDH-B over the first time 10 min is notable. LDH-A preferentially catalyzes pyruvate reduction and NADH oxidation, whereas LDH-B prefers the reverse reaction. Because enhanced NADH oxidation involves NADH binding, it makes sense that LDH-A would bind NADH better.
HOCl- and 1O2-treated LDH-B samples were assayed to determine if oxidant damage affected their ability to enhance NADH oxidation. Once LDH-B was oxidized by 1O2 or HOCl, the rate of NADH oxidation decreased relative to control LDH-B. This was the expected result and further supports the hypothesis that HOCl and 1O2 oxidation of LDH-B affects substrate binding.
4. Discussion
Multiple mammalian LDH-A and LDH-B enzymes, including human, pig, rabbit, and mouse, contain five cysteines with identical or highly conserved flanking sequences [
53]. Our previous work on the ROS oxidation of rabbit muscle LDH-A and, now, on pig heart LDH-B are highly relevant because of these sequence similarities. Pig heart LDH-B and its human and mouse counterparts share 97% sequence homology [
7]. Pig heart LDH-B is 76% identical to its muscle LDH-A [
7]. The enhanced NADH oxidation, in the absence of pyruvate, that we observed with rabbit LDH-A and, herein, with LDH-B (
Figure 6), has also been reported for human, porcine, and bovine LDH enzymes [
27,
33,
34,
35].
Given these similarities, our primary goal was to compare the susceptibility of pig heart LDH-B to ROS oxidation and inhibition with that of LDH-A. Our focus was on the oxidants
1O
2 and HOCl, because of all oxidants tested, those were the only ones that oxidized and inhibited LDH-A [
27]. Herein, multiple techniques including SDS-PAGE under-reducing and nonreducing conditions, cysteine labeling with a fluorescent tag, native gels with activity stain, and kinetic assays were performed to understand LDH-B oxidation and inhibition.
LDH-B, like LDH-A, was inhibited by
1O
2 generated photochemically with multiple photosensitizers, red light, and ambient oxygen (
Figure 1B,C,
Supplemental Figure S1). Higher MW crosslinks indicative of dimers and trimers were observed by SDS-PAGE under reducing conditions (
Figure 1A and
Figure 2). For LDH-A, we also observed a laddering pattern beyond trimers, which is expected for protein tetramers. Although higher MW species in LDH-B were detected, we cannot rule out the formation of crosslinks or individual amino acid oxidation events within a subunit.
Multiple experiments confirm that the oxidant responsible for LDH-B inhibition is
1O
2. The inclusion of D
2O increased LDH-B inhibition (
Figure 1B) because it stabilizes
1O
2 [
39]. Sodium azide is a well-characterized and specific
1O
2 scavenger that protected LDH-B, as shown in
Supplemental Figure S3 [
49] and
Table 1. This is particularly important because photodynamic therapy, which generates
1O
2, is employed to target tumors where LDH enzymes are overexpressed [
25].
The photosensitizer concentrations and irradiation times used to produce
1O
2 and oxidize LDH-B were identical to those employed in our LDH-A work. For this reason, we assert that LDH-A and LDH-B are equally susceptible to oxidation and inhibition by
1O
2. Further, we observed the concurrent inhibition of the two LDH enzymes using our native gel assay with activity stain (
Supplemental Figure S3). When equimolar LDH-A and LDH-B were combined with MB and irradiated, LDH-B was inhibited to the same extent as when LDH-A was not present.
Although cysteine is not the primary amino acid target of
1O
2, we used a cysteine specific fluorescent tag to monitor changes in LDH-B conformation following
1O
2 damage. Pamp et al. reported a conformational change induced by the copper-mediated oxidation of LDH enzymes, which increased cysteine availability [
41]. As for LDH-A, we used a maleimide-based tag, M5F, because Holbrook et al. reported that LDH cysteines react more readily with maleimides than with iodoacetamides [
30].
As anticipated,
1O
2 did not oxidize cysteines, as evidenced by the M5F labeling of the higher MW species in
Figure 2. The quantitation of fluorescein labeling showed that there was no change in the total labeling of LDH-B monomer plus crosslinks, at all times/doses of
1O
2 tested. This is in contrast to LDH-A, where we saw an increase in cysteine labeling at 30 s but not at the longer times (indicative of higher
1O
2 doses). Nonetheless, LDH-A and LDH-B were similar in their extent of M5F labeling. Of the 5 mol cys per mol LDH-B, 2.1 were labeled by M5F (
Figure 5B), whereas, for LDH-A, it was 2.7 [
27].
Our competition studies of
1O
2 damage to LDH-B using different photosensitizers highlight the importance of their chemical properties. When the chlorophyll metabolites, pheoA or chlorin e6, were used rather than MB, higher concentrations of ascorbate and HCA, a catechol, were required to protect LDH-B from oxidation and inhibition by
1O
2 (
Figure 3). Our hypothesis that pheoA and chlorin e6 associate with LDH-B and, therefore, produce
1O
2 in closer proximity to the target amino acids is supported by these results. MB is more water soluble and is charged at physiological pH. This is important because Photofrin is an FDA-approved photosensitizer for photodynamic therapy and its structure resembles that of the chlorophyll metabolites [
54]. Further, a recent review evaluated chlorin e6 for use as a photosensitizer in cancer nanomedicine [
55].
Table 1 summarized our
1O
2 competition studies using ascorbate, HCA, GSH, and EDTA with MB as the photosensitizer. We chose to highlight EDTA because it is a common buffer component—often at millimolar concentrations. EDTA, a tertiary amine, reacts with
1O
2 to produce the EDTA radical cation and superoxide anion [
43,
48]. Its capacity to scavenge
1O
2 is not widely known; consequently, our work cautions that experiments done with
1O
2 in a biological context must consider EDTA reactivity [
48].
Lower concentrations of those scavengers with larger
1O
2 rate constants such as ascorbate and HCA were required to protect 10 μM LDH-B. Higher-than-expected GSH concentrations were required to protect LDH-B based on its comparable rate constant. Because GSH is a charged tripeptide, it is expected to be soluble and, therefore, effective at scavenging
1O
2 generated in solution. Its greater size or association with LDH-B may explain this disparity in
Table 1.
Our results herein, using HOCl as the LDH-B oxidant, diverged to a greater extent from those obtained with LDH-A. While HOCl oxidized LDH-B cysteines and inhibited activity, higher HOCl concentrations (150 μM here vs. 100 μM for LDH-A) were required to see the same extent of inhibition (
Figure 4). We did not observe time-dependent changes in LDH-B conformation that were detected as SDS-PAGE band shifts with HOCl-oxidized LDH-A. We also did not see a LDH-B native gel mobility shift (
Figure 4A) as we did for LDH-A [
27].
Although cysteine is a likely target for oxidation by HOCl, methionine and tyrosine are also oxidized by HOCl [
51]. A comparison of the rabbit muscle LDHA (pdb ID = 5NQQ) with that of pig heart LDH-B (pdb ID = 5YTA) shows that both contain five cysteines, seven tyrosines, and 10 methionines. With LDH-A, we saw a prominent, nonreducible crosslink that was characteristic of dityrosine [
56]. In
Figure 4B, little to no higher MW dimer was visible for HOCl-treated LDH-B in the plus βME lanes. This does not rule out the presence of dityrosine within a subunit, but there was no evidence for that in LDH-B. Under basic conditions, dityrosine is fluorescent and no HOCl-induced fluorescence under basic conditions in LDH-B was observed.
Despite no readily observed conformational changes in LDH-B, oxidation and inhibition by HOCl was not reversed by disulfide-reducing agents. This is consistent with our LDH-A results but differs from our work on HOCl oxidation of tubulin. Disulfides in tubulin formed readily, but 90% of the activity that was lost due to HOCl oxidation was reversed by DTT or TCEP [
37]. Like LDH-A, LDH-B was resistant to oxidation by H
2O
2, chloramines, and Angeli’s salt, a nitroxyl donor that targets cysteines. The millimolar H
2O
2 concentrations tested would have inhibited other proteins we have studied, especially the glycolytic enzyme, GAPDH [
57].
The importance of LDH cysteine oxidation/modification was reinforced decades ago by several researchers who first identified an essential cysteine in both LDH-A and LDH-B. This cysteine in LDH-B is cys164 (in 5YTA) but was cited as cys165 in some early reports [
30]. Modification of it inhibited LDH activity; based on X-ray structures of LDH-B, the nicotinamide ring of NAD
+/NADH is in close proximity. A figure containing two of the four LDH-B subunits was generated from the 5YTA X-ray structure in which NAD and the five cysteines per subunit are highlighted (
Supplemental Figure S5).
We showed that both N-ethyl maleimide and M5F, the fluorescein analog, inhibited LDH-B activity. Therefore, when LDH-B labeling decreased after HOCl oxidation, it is likely that this essential cysteine was oxidized.
Lastly, the ability of LDH-B to enhance NADH oxidation, in the absence of pyruvate, is potentially important in the context of oxidative stress in cancer cells. We were the first to show that
1O
2 could initiate the superoxide-dependent chain reaction that depletes NADH [
27].
Figure 6 shows that LDH-B is comparable to LDH-A in this reaction sequence. Once LDH-B is oxidized and inhibited by either
1O
2 or HOCl, it has the ability to increase NADH oxidation. This is further evidence that these oxidants affect NADH binding by LDH-B.
When photodynamic therapy generates
1O
2, the resulting Type I photo-oxidation yields superoxide anion when hydrogen atom abstraction generates NAD• or amino acid radicals that react with O
2 [
58]. Even if LDH enzymes were not a primary target for ROS, their ability to oxidize NADH and generate H
2O
2 are important mechanisms to affect viability regardless of the cell type. The methodology employed and the results presented about LDH-B oxidation and inhibition are important because LDH enzymes are targets for anti-cancer drug development due to their overexpression and metabolic role in several cancers [
3,
4,
16].