Ascorbate Reacts with Singlet Oxygen to Produce Hydrogen Peroxide
单线态氧是一种高反应性的亲电子物质，它与富含电子的部分迅速反应，例如脂质，硫醇和抗坏血酸（AscH-）的双键。抗坏血酸盐与单线态氧的反应很快（k = 3×108M-1 s-1）。
反应的反应物（1 10 2 + 1AscH-→1H 2 O 2 + 1脱氢山梨酸）的化学计量为1：1。 H2O2的形成导致非常不同的氧化剂，其具有更长的寿命和更大的扩散距离。因此，在生物环境中局部产生的半衰期为1ns-1μs的单线态氧被改变为具有更长寿命的氧化剂，因此可以扩散到远处的目标以引发生物氧化。
单线态氧（1O2)是一种高反应性，亲电子物质，可与富含电子的部分快速反应，如脂质[1,2]，硫醇[3,4]和抗坏血酸的双键[5,6,7,8,9 ]。我们之前已观察到，在中性溶液中，单线态氧与抗坏血酸（AscH-）反应生成H2O2 。该初步研究清楚地表明，在血卟啉衍生物和AscH-的照射系统中的氧消耗是1O2与AscH-反应的结果。
KQ = 3.2×108M-1S-1 [7,8]
O21 + AscH- + H +→H 2 O 2 + DHA
使用Apollo 4000自由基分析仪（World Precision Instruments，Sarasota，FL）同时在搅拌的WPI-NOCHM-4四端口封闭室中同时测量反应混合物的H 2 O 2和O 2浓度。该腔室由透明塑料制成，允许可见光透射到样品。氧电极的校准基于以下假设：离子强度≈85mM的充气水性缓冲液在23-25°C时具有约250μM的氧浓度，并且在用氩气吹扫后具有氧浓度大约0.0μM。过氧化氢电极（WPI，Sarasota，FL）使用推注添加H2O2溶液到pH6.5的无金属磷酸盐缓冲溶液（PBS，50mM）中进行校准。为了除去偶然的催化金属，用螯合树脂（Sigma Chemical Co.St.Louis，MO）处理PBS 。证实了没有金属。使用ε240= 39.4M-1cm-1测定H 2 O 2标准溶液的浓度。 H2O2电极具有较小的背景电流，图11和图2中没有减去背景电流，当确定Δ[H2O2]时，它会下降。因为所有结果都依赖于Δ[H2O2]，这不会影响我们的结果。使用如中的二酸（AscH2，pKa = 4.2）制备抗坏血酸的储备溶液（10mM）;在使用ε265= 14,500M-1cm-1的单阴离子（AscH-）的稀释后，在chelexed PBS（50mM; pH = 7.4）中稀释后验证浓度。
单线态氧与抗坏血酸反应以1：1的化学计量产生H 2 O 2
这些图显示了在无金属磷酸盐缓冲液（pH6.5）中用抗坏血酸盐（1mM）和Photofrin®（225μgmL-1）实时同时测量氧消耗（1）和过氧化氢产生（2）。可见光（hν; 350 J m-2 s-1）引发氧消耗并产生H 2 O 2。如果在对照组中观察到任何氧气消耗很少（3）： 对照组：在没有光，Photofrin或抗坏血酸，或光但没有Photofrin的情况下;当然没有形成H2O2。
叠氮化钠(Sodium azide, NaN 3)在抗坏血酸的光氧化过程中抑制了氧的消耗和H2O2的产生
这些图显示了在pH 6.5的无金属磷酸盐缓冲液中，使用抗坏血酸盐（1 mM）和Photofrin®（225μgmL-1）实时同时测量氧气消耗和过氧化氢产量。可见光（hν，350 J m-2 s-1）引发氧消耗（1,2）和产生H 2 O 2（3,4）。曲线2,4的箭头指示添加NaN 3（1mM）。如图所示，该添加产生了对H2O2电极的干扰，而O2-电极没有观察到这种干扰。然而，结果基于添加叠氮化物之前和之后的曲线的线性部分的斜率。所有溶液都是空气饱和的。
使用来自钨灯泡的可见光完成光敏化实验，所述钨灯泡聚焦以在腔室的中心提供350Jm-2s-1的光强度。使用Yellow Springs Instrument Model 65-A辐射计测量光强度。溶液由Chetric处理的PBS（pH 6.5）中的抗坏血酸盐（1.0mM）和Photofrin（225μgmL-1）组成。在典型的5分钟照射期间加热样品<0.5℃。
Photofrin®是血卟啉衍生物的纯化形式;它是一种用于治疗癌症的光敏剂。暴露在光线下会产生单线态氧，这是一种高度亲电的物质，会引发导致细胞死亡的氧化[12,13]。单线态氧很容易与抗坏血酸反应，产生过氧化氢。在这里，我们使用O2和H2O2的同时实时测量重新检查了该反应的化学计量。使用已经用螯合树脂处理的pH 6.5的磷酸盐缓冲液去除偶然的催化金属，抗坏血酸，抗坏血酸加可见光，有或没有光的Photofrin，或Photofrin加抗坏血酸的溶液中几乎没有氧消耗。在黑暗中，如图1所示。然而，当抗坏血酸和Photofrin的溶液暴露在可见光下时，随着H2O2 的产生,O2快速消耗，与抗坏血酸盐Rxn 1对单线态氧的化学猝灭一致。
使用电极系统，我们可以同时测量消耗的O2总量和同一溶液中产生的H2O2。假设没有其他来源的H 2 O 2或吸收O 2，这些量的比例将提供反应的化学计量。从曝光的开始到结束确定每种浓度的变化。对于图1中呈现的实例，O 2浓度的总变化为123μM，而在曝光的约5分钟期间，H 2 O 2产生的相应变化为150μMH2 O 2。这产生0.82 O 2至1 H 2 O 2的化学计量。
由于实验期间条件的变化，随时间的总变化有时可能会低估或高估事件。更可靠的方法是检查初始斜率。在我们的设置中，我们在灯打开后不久确定了数据的近线性部分期间的斜率。 H2O2产生和O2消耗的这些斜率的绝对值反映了两个过程的速率。在七个独立样品中对这些斜率的测量表明几乎相同的值：O2消耗为-25.1±7.6μM/ min，H2O2产生为+ 29.6±9.9μM/ min。因为H2O2的形成速率取决于分子氧的消失速率，所以这些斜率不是独立的。因此，Rxn-1的化学计量的最佳估计将是七个比率的中值，而不是七个实验的平均值的比率14] *。这些比率的中位数和相关标准差为：
Δ[O 2] /ΔtΔ[H 2 O 2]/Δt=0.86±0.13
为了证明单线态氧参与H2O2的生产，我们研究了广泛使用的单线态氧的物质猝灭剂，NaN3的作用[15,16,17]。如果单线态氧参与反应，那么叠氮化物将在抗坏血酸盐（1mM）的光敏氧化过程中减缓氧的消耗和过氧化氢的产生。叠氮化钠（0.20和1 mM）同时以浓度依赖性方式抑制氧消耗和过氧化氢产生，图2. 1O2与AscH-或叠氮化物（我们的实验条件）反应的速率常数为3.2 ×108 M-1 s-1 和5.8×108 M-1 s-1 。 H2O中1O2的衰变速率常数为kH2O 4.4×105s-1 。因此，溶液中任何物质（i）淬灭的单线态氧的分数为：
FI = kiCikH2O +ΣjkjCj
其中j对所有溶质求和。这假设与所有溶质的反应是二阶的，相对于1O2的一阶和相对于任何具有浓度Cj的溶质的一阶。因此，当存在抗坏血酸盐（1.0mM）时，所形成的1O2的42％将与AscH-反应;当AscH-和叠氮化物均以1.0mM存在时，与AscH-反应的部分将降至21％。因此，对于两种速率，即O 2消失和H 2 O 2形成，我们应该看到斜率的绝对值减少约50％。在实验中，如图2所示，添加叠氮化物会降低这些速率。对于氧气消耗，我们观察到斜率的变浅仅为抗坏血酸盐的53-64％，而对于H2O2的产生，斜率下降42-55％。从图2的代表性数据可以理解，在添加叠氮化物之前和之后立即估计斜率存在相当大的不确定性。这些叠氮化物实验的主要目的是排除三重态敏化剂和抗坏血酸之间的I型反应。如果氧消耗的主要机制和随后形成的H 2 O 2是由于抗坏血酸盐Rxn-1（即II型过程）对1 O 2进行化学猝灭，则速率的这些变化在预期的范围内。
Kwon和Foote使用玫瑰红（rose bengal)作为单线态氧的来源检测了在冷甲醇（CD3OD，-85°C）中这种反应的产物。他们发现了两种异构氢过氧化酮的产生，图3.在升温后，观察到脱氢抗坏血酸（DHA）。在水性环境中，我们期望平行反应方案，由于质子和OH-的容易获得，中间体甚至更不稳定。在pH7.4下，抗坏血酸（AscH2，pKa = 4.2）主要作为单阴离子存在（> 99.9％）。与AscH2相比，这种富电子物质与亲电子物质的反应性更强。因此，我们可以预期在碳-3处形成瞬时加合物，然后将氢过氧化物部分重排为C-2。这允许在C-3处形成缩酮并且在C-2处具有缩酮形成的H 2 O 2的释放。
在近中性水溶液中，单线态氧可与抗坏血酸的富电子碳-3反应。该中间体在C-2处具有高电子密度，导致氢过氧基部分的重排，允许在C-3处形成缩酮，导致抗坏血酸盐快速氧化形成脱氢抗坏血酸（以缩酮形式）和H 2 O 2 。当抗坏血酸盐作为二酸存在时，Kwon和Foote观察到碳-2和碳-3加合物，其中C-2-OOH加合物更丰富。由于众所周知的抗坏血酸和DHA的水解反应，中间体的实际性质可能更复杂。由于抗坏血酸根的稳定性，一小部分抗坏血酸可以通过单电子减少单线态氧，形成超氧化物和抗坏血酸自由基。
抗坏血酸盐易于用作单电子和双电子还原剂。我们也可能期望一小部分1O2可被单电子还原形成超氧化物;这是一个非常有利的反应（（ΔE= + 650（O21 / O∙-2） - （+ 280（Asc∙ - / AscH - ））= + 370mV）。与此一致，使用电子顺磁共振我们观察到Photofrin®/抗坏血酸系统照射时抗坏血酸根的浓度增加，类似于之前报道的，数据未显示。任何形成的超氧化物会立即溶解形成H2O2或与抗坏血酸反应也形成H2O2与Asc• - 。一起产生与Rxn 1相同的1：1化学计量。
单线态氧是非常亲电的并且容易与还原剂如抗坏血酸反应。在这里，我们已经证明该反应是由于化学猝灭产生H 2 O 2。化学计量学最可能是1 1O2: 1 H2O2。
Ascorbate Reacts with Singlet Oxygen to Produce Hydrogen Peroxide
Singlet oxygen is a highly reactive electrophilic species that reacts rapidly with electron-rich moieties, such as the double bonds of lipids, thiols, and ascorbate (AscH-). The reaction of ascorbate with singlet oxygen is rapid (k = 3 × 108 M-1 s-1). Here we have investigated the stoichiometry of this reaction. Using electrodes to make simultaneous, real-time measurements of oxygen and hydrogen peroxide concentrations, we have investigated the products of this reaction. We have demonstrated that hydrogen peroxide is a product of this reaction. The stoichiometry for the reactants of the reaction (1 1O2 + 1AscH- → 1H2O2 + 1dehydroacorbic) is 1:1. The formation of H2O2 results in a very different oxidant that has a longer lifetime and much greater diffusion distance. Thus, locally produced singlet oxygen with a half-life of 1 ns - 1 μs in a biological setting is changed to an oxidant that has a much longer lifetime and thus can diffuse to distant targets to initiate biological oxidations.
Keywords: Ascorbate, singlet oxygen, hydrogen peroxide, photosensitization
Singlet oxygen is a highly reactive, electrophilic species that reacts rapidly with electron-rich moieties, such as the double bonds of lipids [1, 2], thiols [3, 4], and ascorbate [5, 6, 7, 8, 9]. We have observed previously that in neutral solutions singlet oxygen reacts with ascorbate (AscH-) to produce H2O2 . This initial work clearly showed that oxygen consumption in an illuminated system of hematoporphyrin-derivative and AscH- was a result of the reaction of 1O2 with AscH-.
The data suggested a stoichiometry of two 1O2 to one H2O2 . However, this work was done before the scientific community was fully aware of the role that trace levels of adventitious metals would have on ascorbate chemistry . The purpose of these experiments was to re-examine the stoichiometry of this reaction using direct, simultaneous measurements of oxygen and hydrogen peroxide.
2. Materials and Methods
An Apollo 4000 Free Radical Analyzer (World Precision Instruments, Sarasota, FL) was used to simultaneously measure in real-time both H2O2 and O2 concentrations of reaction mixtures in a stirred WPI-NOCHM-4 Four-Port Closed Chamber. This chamber is made of clear plastic that allows visible light transmission to the sample. The calibration of the oxygen-electrode was based on the assumption that aerated aqueous buffer with an ionic strength of ≈85 mM has an oxygen concentration of approximately 250 μM at 23-25°C  and after purging with argon has an oxygen concentration of approximately 0.0 μM. The hydrogen peroxide electrode (WPI, Sarasota, FL) was calibrated using bolus additions of an H2O2 solution into metal-free phosphate buffer solution (PBS, 50 mM) at a pH of 6.5. To remove adventitious catalytic metals the PBS was treated with chelating resin (Sigma Chemical Co. St. Louis, MO) . The absence of metals was verified . The concentration of the H2O2 standard solution was determined using ε240 = 39.4 M-1 cm-1. The H2O2 electrode has a small background current that was not subtracted for Figures Figures11 and and2,2, which drops out when determining Δ[H2O2]. Because all results rely on the Δ[H2O2], this did not affect our results. Stock solutions (10 mM) of ascorbic acid were prepared using the di-acid (AscH2, pKa = 4.2) as in ; the concentration was verified after dilution in chelexed PBS (50 mM; pH=7.4) using ε265 = 14,500 M-1 cm-1 for the monoanion (AscH-) .
Singlet Oxygen reacts with ascorbate to produce H2O2 with a 1:1 stoichiometry
These plots show the real-time, simultaneous measurements of oxygen consumption (1) and hydrogen peroxide production (2) with ascorbate (1 mM) and Photofrin® (225 μg mL-1) in metal-free phosphate buffer, pH 6.5. Visible light (hν; 350 J m-2 s-1) initiated oxygen consumption and production of H2O2. Little if any oxygen consumption is observed (3) in controls: In the absence of light, or Photofrin, or ascorbate, or light but no Photofrin; of course no H2O2 is formed.
Sodium azide suppressed oxygen consumption and H2O2 production during photo-oxidation of ascorbate
These plots show the real-time, simultaneous measurements of oxygen consumption and hydrogen peroxide production with ascorbate (1 mM) and Photofrin® (225 μg mL-1) in metal-free phosphate buffer, pH 6.5. Visible light (hν, 350 J m-2 s-1) initiated oxygen consumption (1,2) and production of H2O2 (3,4). The addition of NaN3 (1 mM) is indicated by arrow for the curves 2, 4. As seen, this addition created a disturbance with the H2O2-electrode, while no such disturbance was seen with the O2-electode. However, results are based on the slope of the linear portion of the curves before and after addition of azide. All solutions were air-saturated.
The photosensitization experiments were accomplished using visible light from a tungsten bulb focused to provide a light intensity of 350 J m-2 s-1 at the center of the chamber. Light intensity was measured using a Yellow Springs Instrument Model 65-A radiometer. Solutions consisted of ascorbate (1.0 mM) and Photofrin® (225 μg mL-1) in Chelex-treated PBS, pH 6.5. Heating of the sample during the typical 5 min of illumination was < 0.5°C.
3. Results and Discussion
Photofrin® is a purified form of hematoporphyrin derivative; it is a photosensitizer used in the treatment of cancer. Upon exposure to light it produces singlet oxygen, a highly electrophilic species that initiates oxidations that lead to cell death [12, 13]. Singlet oxygen reacts readily with ascorbate, producing hydrogen peroxide . Here we have re-examined the stoichiometry of this reaction using simultaneous, real-time measurements of O2 and H2O2. Using phosphate buffer, pH 6.5, which had been treated with chelating resin to remove adventitious catalytic metals , there was little or no oxygen consumption in solutions of ascorbate, ascorbate plus visible light, Photofrin with or without light, or Photofrin plus ascorbate in the dark, Figure 1. However, when solutions of ascorbate and Photofrin were exposed to visible light, rapid consumption of dioxygen ensued with simultaneous production of H2O2, consistent with the chemical quenching of singlet oxygen by ascorbate, Rxn 1.
The Stoichiometry of the reaction between AscH- and1O2 is 1:1 Stoichiometry determined from total amount of O2 consumed and H2O2 formed
With the electrode systems we can simultaneously measure both the total amount of O2 consumed and H2O2 produced in the same solution. Assuming no other sources of H2O2 or sinks for O2, the ratio of these amounts will afford the stoichiometry of the reaction. The change in concentration of each was determined from the beginning to the end of light exposure. For the example presented in Figure 1 the total change in O2 concentration was 123 μM while the corresponding change for the production of H2O2 was 150 μM H2O2 during the ≈5 min of the light exposure. This yields a stoichiometry of 0.82 O2 to 1 H2O2.
Stoichiometry from initial Slopes
Total changes over time can sometimes under- or over-estimate events because of changing conditions during an experiment. A more reliable approach is to examine initial slopes. In our setting we determined the slopes during the near linear portions of the data soon after the light was turned on. The absolute values of these slopes for H2O2-production and O2-consumption reflected the rate of the two processes. Measurements of these slopes in seven independent samples demonstrated nearly the same value: -25.1 ± 7.6 μM/min for O2-consumption and +29.6 ± 9.9 μM/min for H2O2-production. Because the rate of formation of H2O2 is dependent on the rate of disappearance of dioxygen, these slopes are not independent. Thus, the best estimation of the stoichiometry of Rxn-1 will be the median of the seven ratios and not the ratio of the average of the seven experiments 14]*. The median and associated standard deviation of these ratios are:
This value is close to 1, indicating the stoichiometry of the Rxn-1 is 1 mole of oxygen consumed and 1 mole of hydrogen peroxide produced.
Azide demonstrates singlet oxygen involvement
To demonstrate that singlet oxygen is involved in this production of H2O2, we investigated the effect of a widely used physical quencher of 1O2, NaN3 [15, 16, 17]. If singlet oxygen is involved in the reaction, then azide will slow both the consumption of oxygen and the production of hydrogen peroxide during the photosensitized oxidation of ascorbate (1 mM). Sodium azide (0.20 and 1 mM) simultaneously suppressed both oxygen-consumption and hydrogen peroxide-production in a concentration-dependent manner, Figure 2. The rate constants for the reaction of 1O2 with AscH- or azide (with our experimental conditions) are 3.2 × 108 M-1 s-1  and 5.8 × 108 M-1 s-1 , respectively. The decay rate constant for 1O2 in H2O is kH2O 4.4 × 105 s-1 . Thus, the fraction of singlet oxygen being quenched by any species (i) in the solution is:
where j is summed over all solutes. This assumes that reactions with all solutes are second-order, first order with respect to 1O2 and first-order with respect to any solute with concentration Cj. Thus, when ascorbate (1.0 mM) is present, 42% of the 1O2 formed will react with AscH-; when both AscH- and azide are present at 1.0 mM, then the fraction reacting with AscH- will fall to 21%. Thus we should see an approximate 50% decrease in the absolute values of the slopes for both the rates, i.e. O2-disappearance and H2O2-formation. In experiments, such as shown in Figure 2, the addition of azide slows these rates. For oxygen consumption we observed a shallowing of the slope to 53-64% of that with only ascorbate, while for the production of H2O2 the slope decreased 42-55%. As can be appreciated from the representative data of Figure 2, there is considerable uncertainty in estimating the slopes immediately before and just after the addition of azide. The primary purpose of these experiments with azide was to rule out a Type I reaction between triplet state sensitizer and ascorbate. These changes in the rates are in the range of what would be anticipated if the dominant mechanism of oxygen consumption and subsequent formation of H2O2 were due to the chemical quenching of 1O2 by ascorbate, Rxn-1, i.e. a Type II process.
Kwon and Foote examined the products this reaction in cold methanol (CD3OD, -85°C) using rose bengal as a source of singlet oxygen . They found the production of two isomeric hydroperoxy ketones, Figure 3. Upon warming, dehydroascorbic (DHA) was observed. In an aqueous environment we would expect a parallel reaction scheme with the intermediates being even less stable due to the ready availability of protons as well as OH-. At pH 7.4, ascorbic acid (AscH2, pKa = 4.2) is present largely as the monoanion (>99.9%). This electron-rich species is much more reactive with electrophiles than AscH2. Thus we might expect formation of a transient adduct at carbon-3, followed by a rearrangement of the hydroperoxide moiety to C-2. This allows ketal formation at C-3 and the release of H2O2 with ketal formation at C-2.
A proposed mechanism for the production of H2O2
In a near neutral aqueous solution, singlet oxygen may react with the electron-rich carbon-3 of ascorbate. This intermediate will have high electron density at C-2, leading to a rearrangement of the hydroperoxyl moiety, allowing ketal formation at C-3, resulting in rapid oxidation of ascorbate to form dehydroascorbic (in the ketal form) and H2O2 . Kwon and Foote observed both the carbon-2 and carbon-3 adduct when ascorbate was present as the di-acid, with the C-2-OOH adduct in greater abundance. The actual nature of the intermediate maybe more complex, due to the well-known hydrolysis reactions of ascorbate and DHA . Because of the stability of the ascorbate radical, a small fraction of ascorbate may reduce singlet oxygen by one-electron, forming superoxide and the ascorbate free radical .
Ascorbate readily serves as both a one-electron and two-electron reducing agent. We might also expect that a small fraction of 1O2 might be reduced by one-electron to form superoxide ; it is a highly favorable reaction ((ΔE=+650(O21/O∙−2)−(+280(Asc∙−/AscH−))=+370mV) . Consistent with this, using electron paramagnetic resonance we observed an increase in the concentration of ascorbate radical upon illumination of a Photofrin®/ascorbate system, similar to what has been reported previously , data not shown. Any superoxide formed would immediately dismute to form H2O2 or react with ascorbate also forming H2O2 along with Asc•−. This would result in the same 1:1 stoichiometry as with Rxn 1.
Singlet oxygen is very electrophilic and reacts readily with reducing agents, such as ascorbate. Here we have demonstrated that this reaction is due to chemical quenching yielding H2O2. The stoichiometry is most likely 1 1O2: 1 H2O2.
The early observation of this reaction appears to have underestimated the amount of H2O2 produced . That study was done before the importance of adventitious catalytic metals in reactions of ascorbate and H2O2 was fully appreciated by the scientific community. The combination of redox active catalytic metals and ascorbate will both produce and destroy H2O2 . An additional consideration, is that catalase was used as a tool to estimate the amount of H2O2 formed in the system. Catalase compound-I reacts readily with ascorbate and will not return oxygen . The high level of ascorbate in the early experiments would also contribute to an underestimate of total amount of H2O2 generated.
The very fast reaction of ascorbate with singlet oxygen and its high concentration in the water space of cells suggests that it could be an important sink for 1O2 in vivo. The product of the reaction is H2O2, another oxidant. However, there are several enzyme systems that can remove H2O2. This includes catalase, glutathione peroxidase, and the peroxiredoxins. Thus, this additional H2O2 can be detoxified, whereas there are no enzymes systems that directly act on 1O2 in a beneficial way. That AscH- reacts with 1O2 to produce H2O2 stoichiometrically will help in understanding the biological consequences of generation of singlet oxygen.
Photochem Photobiol. Author manuscript; available in PMC 2007 Dec 18.
Published in final edited form as:
Photochem Photobiol. 2006; 82(6): 1634–1637.
Galina G. Kramarenko, Stephen G. Hummel, Sean M. Martin, and Garry R. Buettner*
Free Radical and Radiation Biology and The University of Iowa, Iowa City, IA 52242-1101
Ascorbate Reacts with Singlet Oxygen to Produce Hydrogen Peroxide https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2147043/