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Article

Lactate Dehydrogenase-B Oxidation and Inhibition by Singlet Oxygen and Hypochlorous Acid

by
Lisa M. Landino
* and
Emily E. Lessard
Department of Chemistry, College of William & Mary, Williamsburg, VA 23187-8795, USA
*
Author to whom correspondence should be addressed.
Oxygen 2024, 4(4), 432-448; https://doi.org/10.3390/oxygen4040027
Submission received: 27 October 2024 / Revised: 17 November 2024 / Accepted: 21 November 2024 / Published: 24 November 2024
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Abstract

:
Alterations in cellular energy metabolism are a hallmark of cancer and lactate dehydrogenase (LDH) enzymes are overexpressed in many cancers regardless of sufficient oxygen and functional mitochondria. Further, L-lactate plays signaling roles in multiple cell types. We evaluated the effect of singlet oxygen and hypochlorous acid (HOCl) on pig heart LDH-B, which shares 97% homology with human LDH-B. Singlet oxygen was generated photochemically using methylene blue or the chlorophyll metabolites, pheophorbide A and chlorin e6. Singlet oxygen induced protein crosslinks observed by SDS-PAGE under reducing conditions and inhibited LDH-B activity. Ascorbate, hydrocaffeic acid, glutathione and sodium azide were employed as singlet oxygen scavengers and shown to protect LDH-B. Using fluorescein-modified maleimide, no changes in cysteine availability as a result of singlet oxygen damage were observed. This was in contrast to HOCl, which induced the formation of disulfides between LDH-B subunits, thereby decreasing LDH-B labeling with fluorescein. HOCl oxidation inhibited LDH-B activity; however, disulfide reduction did not restore it. LDH-B cysteines were resistant to millimolar H2O2, chloramines and Angeli’s salt. In the absence of pyruvate, LDH-B enhanced NADH oxidation in a chain reaction initiated by singlet oxygen that resulted in H2O2 formation. Once damaged by either singlet oxygen or HOCl, NADH oxidation by LDH-B was impaired.

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 (1O2) induced the formation of multiple crosslinks between LDH-A subunits, thereby inhibiting its activity [24]. We were particularly interested in 1O2 because it is the primary oxidant produced during photodynamic therapy [25,26]. We also investigated the effects of 1O2 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 1O2, HOCl, and H2O2 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 1O2. Its susceptibility to oxidation and inhibition by 1O2 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 H2O2, 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 (O2•−) that yields H2O2. Wu et al. demonstrated in HeLa cells that both LDH-A and LDH-B were responsible for increased H2O2 production and overall oxidative stress [35]. Herein, we show that 1O2 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 (ε292 = 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 1O2 concentration.

3.1. Photo-Oxidation of LDH-B

To test the susceptibility of porcine LDH-B to oxidation by 1O2, 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% D2O were also assayed for activity because D2O increases the reactivity of 1O2 by extending its lifetime. This is attributed to the lower-frequency vibrational stretching mode of D2O relative to H2O [39]. At all times tested, the inclusion of 20% D2O increased the extent of LDH-B inhibition (Figure 1B). D2O 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 1O2 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 1O2 [40]. Nonetheless, we were interested in changes to LDH-B cysteine availability as a result of 1O2 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 1O2. As the dose of red light and, consequently, 1O2 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, 1O2 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 1O2 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 1O2, we performed competition studies using well-characterized 1O2 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 1O2 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 1O2 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 1O2 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 1O2 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 1O2 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 1O2 scavenger, but our recent work showed that it reacts with 1O2 and produces H2O2 [43,44]. EDTA is a common buffer component; therefore, it is imperative to assess its ability to scavenge 1O2.
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 1O2 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 1O2. EDTA reacts with 1O2 to generate the EDTA radical cation and superoxide anion with a rate constant of ~105 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 1O2 scavenger comparable to HCA, though less reactive than ascorbate (Table 1).
We also tested sodium azide (NaN3) for its ability to protect LDH-B from 1O2 because it is a well-established and specific 1O2 scavenger [47,49]. Supplemental Figure S3 shows that 0.1 mM to 1 mM NaN3 protected LDH-B activity as assessed by native gel with activity stain. Protection by NaN3 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 1O2 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 (H2O2) did not inhibit LDH-B activity at concentrations up to 5 mM, nor did it induce formation of disulfides. This resistance to H2O2 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 1O2 (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 1O2 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 1O2 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 1O2. The inclusion of D2O increased LDH-B inhibition (Figure 1B) because it stabilizes 1O2 [39]. Sodium azide is a well-characterized and specific 1O2 scavenger that protected LDH-B, as shown in Supplemental Figure S3 [49] and Table 1. This is particularly important because photodynamic therapy, which generates 1O2, is employed to target tumors where LDH enzymes are overexpressed [25].
The photosensitizer concentrations and irradiation times used to produce 1O2 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 1O2. 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 1O2, we used a cysteine specific fluorescent tag to monitor changes in LDH-B conformation following 1O2 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, 1O2 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 1O2 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 1O2 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 1O2 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 1O2 (Figure 3). Our hypothesis that pheoA and chlorin e6 associate with LDH-B and, therefore, produce 1O2 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 1O2 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 1O2 to produce the EDTA radical cation and superoxide anion [43,48]. Its capacity to scavenge 1O2 is not widely known; consequently, our work cautions that experiments done with 1O2 in a biological context must consider EDTA reactivity [48].
Lower concentrations of those scavengers with larger 1O2 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 1O2 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 H2O2, chloramines, and Angeli’s salt, a nitroxyl donor that targets cysteines. The millimolar H2O2 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 1O2 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 1O2 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 1O2, the resulting Type I photo-oxidation yields superoxide anion when hydrogen atom abstraction generates NAD• or amino acid radicals that react with O2 [58]. Even if LDH enzymes were not a primary target for ROS, their ability to oxidize NADH and generate H2O2 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].

5. Conclusions

Our results summarize the effects of ROS on pig heart LDH-B, which shares 97% homology with its human counterpart. Of all ROS tested, only 1O2 and HOCl inhibited LDH-B activity and induced the formation of covalent crosslinks between subunits of the homotetramer. This is consistent with our prior work on mammalian LDH-A. LDH-B activity was protected from 1O2 by multiple scavengers including sodium azide, ascorbate, the catechol HCA, and GSH. HOCl induced the formation of interchain disulfides resulting in a dose-dependent loss of LDH-B activity; however, their reduction did not restore LDH-B function. Lastly, LDH-B catalyzed NADH oxidation in the absence of pyruvate in a manner similar to LDH-A. Once LDH-B was irreversibly damaged by 1O2 or HOCl, it could not catalyze this reaction. This work is valuable because LDH enzymes are often overexpressed in many cancers.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/oxygen4040027/s1, Figure S1: Native gel electrophoresis of LDH-B with three photosensitizers; Figure S2: SDS-PAGE of LDH-B treated with three photosensitizers; Figure S3: Protection of LDH-B from 1O2 by sodium azide; Figure S4: Native gel electrophoresis of LDH-A and LDH-B; Figure S5: Structure of LDH-B with NAD and cysteines highlighted.

Author Contributions

L.M.L.:conceptualization, methodology, investigation, formal analysis, writing—original draft preparation, writing—review and editing, supervision, project administration. E.E.L.: methodology, investigation, figure preparation. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Raw data, procedures, and all Excel files are available upon request.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

DTT, dithiothreitol; GSH, glutathione; HOCl, hypochlorous acid; HCA, hydrocaffeic acid; LDH, lactate dehydrogenase; MB, methylene blue; M5F, fluorescein-5-maleimide; MPO, myeloperoxidase; NBT, nitroblue tetrazolium; NaN3, sodium azide; PB, phosphate buffer; pheoA, pheophorbide A; PMS, phenazine methosulfate; SOD, superoxide dismutase; TCEP, Tris(2-carboxyethyl)phosphine

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Figure 1. 1O2 oxidation of LDH-B. LDH-B (10 µM) samples containing 1 µM MB were irradiated for 0–90 s. (A) Samples were analyzed by SDS-PAGE under reducing conditions on 10% gels and stained with Coomassie blue. (B) LDH-B samples were prepared as in (A). After irradiation, samples were diluted 1:10 with 20 mM Tris pH 8.8. Kinetic assays (200 mL) contained 5 µL 1:10 LDH-B, 4 mM lithium lactate and 2 mM NAD+ in 20 mM Tris pH 8.8. Data shown is the average of at least three independent experiments performed in duplicate. NAD+ reduction was monitored at 340 nm in a 96-well plate at 30 °C for 4 min. (C) LDH-B samples prepared as in A were analyzed by native gel electrophoresis on 0.8% agarose gels. Activity stain contained 0.75 mM NAD+, 25 mM lithium lactate, 8–10 mg NBT, and 1–2 mg PMS in 100 mM Tris pH 8.6.
Figure 1. 1O2 oxidation of LDH-B. LDH-B (10 µM) samples containing 1 µM MB were irradiated for 0–90 s. (A) Samples were analyzed by SDS-PAGE under reducing conditions on 10% gels and stained with Coomassie blue. (B) LDH-B samples were prepared as in (A). After irradiation, samples were diluted 1:10 with 20 mM Tris pH 8.8. Kinetic assays (200 mL) contained 5 µL 1:10 LDH-B, 4 mM lithium lactate and 2 mM NAD+ in 20 mM Tris pH 8.8. Data shown is the average of at least three independent experiments performed in duplicate. NAD+ reduction was monitored at 340 nm in a 96-well plate at 30 °C for 4 min. (C) LDH-B samples prepared as in A were analyzed by native gel electrophoresis on 0.8% agarose gels. Activity stain contained 0.75 mM NAD+, 25 mM lithium lactate, 8–10 mg NBT, and 1–2 mg PMS in 100 mM Tris pH 8.6.
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Figure 2. Cysteine labeling of LDH-B with fluorescein. LDH-B (10 μM) samples containing 1 μM MB were irradiated for 0–120 s. Samples were treated with 500 μM M5F for 30 min at 37 °C and analyzed by SDS-PAGE under reducing conditions on 10% gels. Fluorescent images were captured using a Bio-Rad Chemi-doc XRS imaging system (left). After imaging, gels were stained with Coomassie blue (right).
Figure 2. Cysteine labeling of LDH-B with fluorescein. LDH-B (10 μM) samples containing 1 μM MB were irradiated for 0–120 s. Samples were treated with 500 μM M5F for 30 min at 37 °C and analyzed by SDS-PAGE under reducing conditions on 10% gels. Fluorescent images were captured using a Bio-Rad Chemi-doc XRS imaging system (left). After imaging, gels were stained with Coomassie blue (right).
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Figure 3. LDH-B protection from 1O2 by ascorbate and HCA. (A) LDH-B (10 μM) samples containing 1 μM MB were irradiated for 120 s. Ascorbate (100 or 250 μM) was added prior to irradiation. Samples were analyzed by SDS-PAGE under reducing conditions on 10% gels and stained with Coomassie blue. (B) LDH-B (10 μM) samples containing 1 μM MB or 15 μM pheoA were irradiated for 120 s. Ascorbate (250 or 500 μM) was added prior to irradiation. “D” indicates dark. Samples were analyzed by native gel electrophoresis on 0.8% agarose gels. Activity stain contained 0.75 mM NAD+, 25 mM lithium lactate, 8–10 mg NBT, and 1–2 mg PMS in 100 mM Tris pH 8.6. (C) LDH-B samples contained 1 μM MB, 2.5 μM chlorin e6, or 15 μM pheoA. Either 0.5 mM ascorbate or 1.5 mM HCA was added prior to irradiation. LDH-B activity was measured as in Figure 1B.
Figure 3. LDH-B protection from 1O2 by ascorbate and HCA. (A) LDH-B (10 μM) samples containing 1 μM MB were irradiated for 120 s. Ascorbate (100 or 250 μM) was added prior to irradiation. Samples were analyzed by SDS-PAGE under reducing conditions on 10% gels and stained with Coomassie blue. (B) LDH-B (10 μM) samples containing 1 μM MB or 15 μM pheoA were irradiated for 120 s. Ascorbate (250 or 500 μM) was added prior to irradiation. “D” indicates dark. Samples were analyzed by native gel electrophoresis on 0.8% agarose gels. Activity stain contained 0.75 mM NAD+, 25 mM lithium lactate, 8–10 mg NBT, and 1–2 mg PMS in 100 mM Tris pH 8.6. (C) LDH-B samples contained 1 μM MB, 2.5 μM chlorin e6, or 15 μM pheoA. Either 0.5 mM ascorbate or 1.5 mM HCA was added prior to irradiation. LDH-B activity was measured as in Figure 1B.
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Figure 4. LDH oxidation and inhibition by HOCl. LDH-B (10 μM) in 10 mM PB pH 7.4 was treated with up to 150 μM HOCl for 30 min at 30 °C. (A) Samples were analyzed by native gel electrophoresis on 0.8% agarose gels. Activity stain contained 0.75 mM NAD+, 25 mM lithium lactate, 8–10 mg NBT, and 1–2 mg PMS in 100 mM Tris pH 8.6. (B) Samples were analyzed by SDS-PAGE under reducing and nonreducing conditions (±βME). The gel was stained with Coomassie Blue. (C) Kinetic assays (200 μL) contained 5 μL 1:10 LDH-B, 4 mM lithium lactate, and 2 mM NAD+ in 20 mM Tris pH 8.8. Data shown is the average of at least three independent experiments performed in duplicate. NAD+ reduction was monitored at 340 nm in a 96-well plate at 30 °C for 4 min.
Figure 4. LDH oxidation and inhibition by HOCl. LDH-B (10 μM) in 10 mM PB pH 7.4 was treated with up to 150 μM HOCl for 30 min at 30 °C. (A) Samples were analyzed by native gel electrophoresis on 0.8% agarose gels. Activity stain contained 0.75 mM NAD+, 25 mM lithium lactate, 8–10 mg NBT, and 1–2 mg PMS in 100 mM Tris pH 8.6. (B) Samples were analyzed by SDS-PAGE under reducing and nonreducing conditions (±βME). The gel was stained with Coomassie Blue. (C) Kinetic assays (200 μL) contained 5 μL 1:10 LDH-B, 4 mM lithium lactate, and 2 mM NAD+ in 20 mM Tris pH 8.8. Data shown is the average of at least three independent experiments performed in duplicate. NAD+ reduction was monitored at 340 nm in a 96-well plate at 30 °C for 4 min.
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Figure 5. Cysteine labeling after HOCl oxidation. LDH-B (10 μM) in 10 mM PB pH 7.4 was treated with up to 150 μM HOCl for 30 min at 30 °C. Excess HOCl was quenched with 0.2 mM S-methyl-cys. (A) Samples were treated with 500 μM M5F for 30 min at 37 °C and analyzed by SDS-PAGE under nonreducing conditions on 10% gels. Fluorescent images were captured using a Bio-Rad Chemi-doc XRS imaging system (left). After imaging, gels were stained with Coomassie blue (right). (B) Samples were prepared as in (A) except reactions were 60 μL. M5F-labeled LDH-B was precipitated with 80% ethanol. Fluorescein was quantitated at 495 nm relative to a fluorescein standard curve. These data are the average of two independent trials performed in duplicate.
Figure 5. Cysteine labeling after HOCl oxidation. LDH-B (10 μM) in 10 mM PB pH 7.4 was treated with up to 150 μM HOCl for 30 min at 30 °C. Excess HOCl was quenched with 0.2 mM S-methyl-cys. (A) Samples were treated with 500 μM M5F for 30 min at 37 °C and analyzed by SDS-PAGE under nonreducing conditions on 10% gels. Fluorescent images were captured using a Bio-Rad Chemi-doc XRS imaging system (left). After imaging, gels were stained with Coomassie blue (right). (B) Samples were prepared as in (A) except reactions were 60 μL. M5F-labeled LDH-B was precipitated with 80% ethanol. Fluorescein was quantitated at 495 nm relative to a fluorescein standard curve. These data are the average of two independent trials performed in duplicate.
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Figure 6. Enhanced NADH oxidation by LDH-A and LDH-B: activation by 1O2. ll reactions (100 μL) contained 200 μM NADH in 50 mM PB pH 7.1 in a 96-well plate. 1O2 formation was initiated with 2.5 μM MB and 10 s of red light. Additions included LDH-A or LDH-B (10 μM). Absorbance at 340 nm was measured prior to and immediately after light exposure. These data represent the average of two independent experiments performed in triplicate.
Figure 6. Enhanced NADH oxidation by LDH-A and LDH-B: activation by 1O2. ll reactions (100 μL) contained 200 μM NADH in 50 mM PB pH 7.1 in a 96-well plate. 1O2 formation was initiated with 2.5 μM MB and 10 s of red light. Additions included LDH-A or LDH-B (10 μM). Absorbance at 340 nm was measured prior to and immediately after light exposure. These data represent the average of two independent experiments performed in triplicate.
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Table 1. Effect of 1O2 scavengers on LDH-B activity.
Table 1. Effect of 1O2 scavengers on LDH-B activity.
Singlet Oxygen Scavenger% Control ActivityRate Constant
(M−1 s−1)
Dark100
Light (120 s)25 ± 5
Light + 10 mM ascorbate40 ± 103 × 108 [45]
Light + 50 mM ascorbate 78 ± 5
Light + 50 mM HCA57 ± 45.5 × 108 [46]
Light + 500 mM HCA97 ± 6
Light + 100 mM GSH55 ± 32 × 108 [46]
Light + 1 mM GSH83 ± 6
Light + 0.25 mM EDTA45 ± 10~105 [43]
Light + 1 mM EDTA84 ± 3
Light + 1 mM sodium azide85 ± 5~109 [47]
LDH-B (10 μM) and 1 μM MB in 10 mM PB pH 7.4 was combined with each 1O2 scavenger prior to irradiation. LDH-B activity assays were performed as described in Figure 1B.
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Landino, L.M.; Lessard, E.E. Lactate Dehydrogenase-B Oxidation and Inhibition by Singlet Oxygen and Hypochlorous Acid. Oxygen 2024, 4, 432-448. https://doi.org/10.3390/oxygen4040027

AMA Style

Landino LM, Lessard EE. Lactate Dehydrogenase-B Oxidation and Inhibition by Singlet Oxygen and Hypochlorous Acid. Oxygen. 2024; 4(4):432-448. https://doi.org/10.3390/oxygen4040027

Chicago/Turabian Style

Landino, Lisa M., and Emily E. Lessard. 2024. "Lactate Dehydrogenase-B Oxidation and Inhibition by Singlet Oxygen and Hypochlorous Acid" Oxygen 4, no. 4: 432-448. https://doi.org/10.3390/oxygen4040027

APA Style

Landino, L. M., & Lessard, E. E. (2024). Lactate Dehydrogenase-B Oxidation and Inhibition by Singlet Oxygen and Hypochlorous Acid. Oxygen, 4(4), 432-448. https://doi.org/10.3390/oxygen4040027

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