Human and rodent red blood cells do not demonstrate xanthine oxidase activity or XO-catalyzed nitrite reduction to NO

Abstract

A number of molybdopterin enzymes, including xanthine oxidoreductase (XOR), aldehyde oxidase (AO), sulfite oxidase (SO), and mitochondrial amidoxime reducing component (mARC), have been identified as nitrate and nitrite reductases. Of these enzymes, XOR has been the most extensively studied and reported to be a substantive source of nitric oxide (NO) under inflammatory/hypoxic conditions that limit the catalytic activity of the canonical NOS pathway. It has also been postulated that XOR nitrite reductase activity extends to red blood cell (RBCs) membranes where it has been immunohistochemically identified. These findings, when combined with countervailing reports of XOR activity in RBCs, incentivized our current study to critically evaluate XOR protein abundance/enzymatic activity in/on RBCs from human, mouse, and rat sources. Using various protein concentrations of RBC homogenates for both human and rodent samples, neither XOR protein nor enzymatic activity (xanthine → uric acid) was detectable. In addition, potential loading of RBC-associated glycosaminoglycans (GAGs) by exposing RBC preparations to purified XO before washing did not solicit detectable enzymatic activity (xanthine → uric acid) or alter NO generation profiles. To ensure these observations extended to absence of XOR-mediated contributions to overall RBC-associated nitrite reduction, we examined the nitrite reductase activity of washed and lysed RBC preparations via enhanced chemiluminescence in the presence or absence of the XOR-specific inhibitor febuxostat (Uloric®). Neither addition of inhibitor nor the presence of the XOR substrate xanthine significantly altered the rates of nitrite reduction to NO by RBC preparations from either human or rodent sources confirming the absence of XO enzymatic activity. Furthermore, examination of the influence of the age (young cells vs. old cells) of human RBCs on XO activity also failed to demonstrate detectable XO protein. Combined, these data suggest: 1) that XO does not contribute to nitrite reduction in/on human and rodent erythrocytes, 2) care should be taken to validate immuno-detectable XO by demonstrating enzymatic activity, and 3) XO does not associate with human erythrocytic glycosaminoglycans or participate in nonspecific binding.

Keywords: Glycosaminoglycans; Nitric oxide; Nitrite; Red blood cells; Xanthine oxidoreductase.

Fig. 1.

Human, mouse, and rat RBCs do not demonstrate XO activity. Human, mouse, and rat plasma (A) and washed RBC homogenates (B) were analyzed for XO activity as described in the methods. The volume of RBC homogenate used was 16 μL which corresponds to 0.8 mg protein. Data represent the mean ± standard deviation, n = 6 for human, n = 3 mice, and n = 4 for rat samples.

Fig. 2.

RBCs do not bind XO. A) Cartoon depiction of XO bound to heparin-Sepharose 6B. B) Purified XO (50 mU) was added to human blood for 15 min and then the samples washed under identical conditions as in Fig. 1 and in the methods. Samples were then assessed for XO enzymatic activity as in Fig. 1. Control samples were exposed to either the XO inhibitor allopurinol (Allo) or febuxostat (Febux) as indicated. Data represent the mean ± standard deviation n = 4.

Fig. 3.

Assessment of RBC-associated XOR protein abundance and impact of cellular age. A) Western blot analysis was performed on human, mouse, and rat RBC homogenates using a monoclonal anti-XOR antibody (Santa Cruz) as described in the methods. Purified XO was used as a positive control and electrophoresed using 0.5 μg total protein. A total of 30 μg of protein was electrophoresed for each homogenate (human, mouse, and rat). Shown is a representative of 3 independent blots representing a total of 3 mice, 3 rats and 3 humans. B) Western blot analysis was performed on five different human RBC homogenates, either young or old, using the same mouse monoclonal anti-XOR antibody as described above. Purified XO was used as a positive control and electrophoresed using 0.5 μg total protein. (HV = healthy volunteer/Y = young/O = old).

Fig. 4.

Human and Rodent RBCs do not demonstrate XO-catalyzed nitrite reductase activity. Human, mouse, and rat samples were analyzed for XOR-dependent contributions to overall NO₂⁻ reductase activity using the Sievers Nitric Oxide Analyzer (NOA) as detailed in the methods. Samples were prepared in the reaction chamber with 50 μL of RBC homogenate (5 mg total protein) in KPi pH 7.0 with or without xanthine (25 μM) and with or without febuxostat (10 μM). Reactions were initiated by the addition of NaNO₂⁻ (500 μM). A) Representative NO generation profiles from human RBC homogenates + xanthine with or without addition of febuxostat (Febux). B) Human, mouse, and rat RBC homogenate values (alone or with xanthine or with xanthine and febuxostat). Authentication of NO was determined by diminution of the signal by the presence of the electron spin resonance NO spin trap carboxy-PTIO. Data represent the mean ± SD of n = 4.