It is proposed that the bond between nitric oxide (NO) and the Hb thiol Cys-␤ 93 (SNOHb) is favored when hemoglobin (Hb) is in the relaxed (R, oxygenated) conformation, and that deoxygenation to tense (T) state destabilizes the SNOHb bond, allowing transfer of NO from Hb to form other (vasoactive) S-nitrosothiols (SNOs). However, it has not previously been possible to measure SNOHb without extensive Hb preparation, altering its allostery and SNO distribution. Here, we have validated an assay for SNOHb that uses carbon monoxide (CO) and cuprous chloride (CuCl)-saturated Cys. This assay is specific for SNOs and sensitive to 2-5 pmol. Uniquely, it measures the total SNO content of unmodified erythrocytes (RBCs) (SNORBC), preserving Hb allostery. In room air, the ratio of SNORBC to Hb in intact RBCs is stable over time, but there is a logarithmic loss of SNORBC with oxyHb desaturation (slope, 0.043). This decay is accelerated by extraerythrocytic thiol (slope, 0.089; P < 0.001). SNORBC stability is uncoupled from O2 tension when Hb is locked in the R state by CO pretreatment. Also, SNORBC is increased Ϸ20-fold in human septic shock (P ‫؍‬ 0.002) and the O2-dependent vasoactivity of RBCs is affected profoundly by SNO content in a murine lung bioassay. These data demonstrate that SNO content and O2 saturation are tightly coupled in intact RBCs and that this coupling is likely to be of pathophysiological significance.sepsis ͉ nitric oxide ͉ vascular physiology E vidence has accumulated for an S-nitrosothiol (SNO)-based vascular signaling system in which hemoglobin (Hb) reactions with nitric oxide (NO) transduce redox gradients into bioactivities (1-6). There is agreement that human Hb undergoes Snitrosylation at Cys-␤ 93 (3,(7)(8)(9)(10)(11). Erythrocytes are proposed to couple O 2 tension to the distribution of NO activities (such as control of blood flow) by linking the allosteric transition of Hb (12, 13) to conformation-dependent changes in the redox activity of this Cys-␤ 93 (13-18) and the stereochemistry of this SNO bond at Cys-␤ 93 (6, 7). Indeed, Cys-␤ 93 SNO in human Hb (SNOHb) can be crystallized only with the Hb tetramer in the relaxed (R, oxygenated) conformation; the SNO bond is unstable with Hb in the tense (T, deoxygenated) conformation (7). These observations support a paradigm in which NO binding to Cys-␤ 93 is favored in the R state and NO binding to Fe(II) (and͞or transnitrosation to an alternate thiol) is favored in the T state (19-21). Thus, the change in stability of Cys-␤ 93 SNO during Hb transition between R and T states may serve to couple regional O 2 gradients to the deployment or quenching of NO bioactivities in the microcirculation (2, 6, 22).However, assaying SNOHb has been problematic. First, detection of the SNO bond has required dilution and͞or pretreatment of Hb to (i) control for artifactual identification of nitrite and Fenitrosyl species and (ii) prevent autocapture of NO on Fe during analysis (8,19,(23)(24)(25). As a result, attempts to quantify Cys-␤ 93 SNO density can be biased ...

Adenovirus is a focus of the water treatment community because of its resistance to standard, monochromatic low-pressure (LP) UV irradiation. Recent research has shown that polychromatic, medium-pressure (MP) UV sources are more effective than LP UV for disinfection of adenovirus when viral inactivation is measured using cell culture infectivity assays; however, UV-induced DNA damage may be repaired during cell culture infectivity assays, and this confounds interpretation of these results. Objectives of this work were to study adenoviral response to both LP and MP UV using (i) standard cell culture infectivity assays and (ii) a PCR assay to directly assess damage to the adenoviral genome without introducing the virus into cell culture. LP and MP UV dose response curves were determined for (i) log inactivation of the virus in cell culture and (ii) UV-induced lesions per kilobase of viral DNA as measured by the PCR assay. Results show that LP and MP UV are equally effective at damaging the genome; MP UV is more effective at inactivating adenovirus in cell culture. This work suggests that the higher disinfection efficacy of MP UV cannot be attributed to a difference in DNA damage induction. These results enhance our understanding of the fundamental mechanisms of UV disinfection of viruses-especially double-stranded DNA viruses that infect humans-and improve the ability of the water treatment community to protect public health.

Adenoviruses are resistant to monochromatic, low-pressure (LP) UV disinfection-but have been shown to be susceptible to inactivation by polychromatic, medium-pressure (MP) UV-when assayed using cell culture infectivity. One possible explanation for the difference between UV lamp types is that the additional UV wavelengths emitted by MP UV enable it to cause greater damage to viral proteins than LP UV. The objective of this study was to examine protein damage in adenoviruses treated with LP and MP UV. Results show that MP UV is more effective at damaging viral proteins at high UV doses, though LP UV caused some damage as well. To our knowledge, this study is the first to investigate protein damage in UV-treated adenovirus, and the overview presented here is expected to provide a basis for further, more detailed work.A significant amount of data has been published on UV inactivation of adenovirus and other viruses using monochromatic low-pressure (LP) UV followed by assays of infectivity using cell culture; these studies have shown adenovirus to be highly resistant to LP UV disinfection (2). Low-pressure UV is understood to inactivate pathogens by damaging their genomes (4, 5). In adenovirus, genomic DNA damage may be repaired in host cells, resulting in its apparent UV resistance. When irradiated with mediumpressure (MP) UV, adenoviruses have been shown to be both more sensitive to inactivation than they are upon irradiation with LP UV and as susceptible to UV inactivation as other viruses, even in standard cell culture infectivity assays (1, 6). Medium-pressure UV is polychromatic-it emits a range of wavelengths in the germicidal portion of the UV emission spectrum (200 to 300 nm) which are absorbed by both DNA and proteins, and so MP UV has the potential to damage adenoviral proteins in addition to the genome. Viral proteins are an integral part of every step in the process of infection and enable adenoviruses to successfully infect host cells even if their DNA is damaged (12). Specific adenoviral proteins and UV damage to proteins have been discussed elsewhere (4,5,11,14). Here we describe a study investigating protein damage in LP and MP UV-treated adenovirus using SDS-PAGE. We hypothesize that MP UV is more effective at causing protein damage than LP UV. This work represents an important first step in this field and will help provide a foundation for further, more detailed work.Preparation of virus, UV irradiation, and dose calculation were carried out as previously described (1). Three independent UV irradiation experiments were conducted for each UV dose; protein precipitation and SDS-PAGE were done twice for each independent experiment. For protein precipitation, 1 ml of irradiated virus was spiked with aprotinin as an internal standard, pretreated with 0.05% sodium deoxycholate, precipitated with 10% trichloroacetic acid (TCA) (9), and resuspended directly in Laemmli sample buffer (Bio-Rad, Hercules, CA). Standard SDS-PAGE was carried out using 4 to 20% gradient Tris-HCl ReadyGel minigels that were fixed...