Stimulation of mouse lymphocytes by a mitogen derived from Mycoplasma arthritidis. IV. Murine T hybridoma cells exhibit differential accessory cell requirements for activation by M. arthritidis T cell mitogen, concanavalin A, or hen egg-white lysozyme

A T-cell mitogen present in culture supernatants of Mycoplasma arthritidis (MAS) is known to exhibit an absolute dependence on E alpha-bearing accessory cells (AC), which appear to function by binding the mitogen. We therefore compared the specificity and nature of the AC requirements for MAS and antigen-induced production of IL 2 in T hybridoma cell lines originating from a fusion by using hen egg-white lysozyme (HEL)-specific, H-2d-restricted T blasts. A marked specificity was noted in the ability of the hybridoma lines to become activated by Con A, MAS, or HEL antigen. Thus all three lines produced IL 2 in response to Con A without the addition of B lymphoma AC. Two lines responded to MAS, but only in the presence of AC, and only one line responded to HEL antigen in the presence of AC. Using the HEL responsive T hybridoma line, we demonstrated that disrupted AC and AC membranes could present MAS but not HEL. MAS rapidly associated with AC at 4 degrees C, whereas HEL failed to do so. Paraformaldehyde-fixed AC could absorb the mitogen in MAS and present it to T hybridoma cells within several minutes, whereas HEL antigen could only be presented by fixed AC if there was a prolonged period of incubation (greater than 30 min) at 37 degrees C before fixation. The combined data indicate that metabolically active cells are not required for the association of MAS with AC or for presentation of MAS to T hybridomas. In contrast, HEL antigen requires metabolically active cells for both of these processes. Thus, the mitogen in MAS can bind to AC without any processing requirements, and it is likely that the resulting complex of mitogen and Ia molecules can directly activate T hybridoma cells.     

Accessory cell function in the Con A response: role of Ia-positive and Ia-negative accessory cells

We have examined the role of Ia-positive and Ia-negative accessory cells (AC) and soluble factors in Con A-stimulated murine T cell activation. Supernatant fluids containing interleukin 1 (IL 1) derived from the P388D1 macrophage cell line and from a lipopolysaccharide (LPS)-stimulated macrophage hybridoma provided only partial reconstitution of the response of purified T cells (18 to 27%). The complete reconstitution obtained with gamma-irradiated spleen cells or LPS-activated B cells was inhibited by approximately 60 to 77% when anti-Ia antibody was included in the culture. Despite this apparent involvement of Ia+ spleen AC, Ia-negative L cell AC could also reconstitute the response of both Class I-restricted Lyt-2+ T cells and Class II-restricted L3T4+ T cells. When the Ia-negative AC were employed, the L3T4 antigen on L3T4+ T cells played a critical role because addition of anti-L3T4 antibody to the culture inhibited the response by 85 to 90%. In contrast, anti-L3T4 did not inhibit the response in the presence of spleen AC. These results suggest that the molecules involved in T cell-AC interactions may vary depending on the AC source. Moreover, at least one of the putative target ligands for L3T4 presumably is not Ia, because anti-L3T4 inhibited T cell stimulation when Ia-negative AC were used.     

人生长分化因子15单克隆抗体制备、鉴定及药用价值初探

目的:制备稳定分泌抗人生长分化因子15(GDF15)单克隆抗体(m Ab)的杂交瘤细胞系,并对其分泌的m Ab进行鉴定。方法:根据人GDF15氨基酸序列特征,设计合成了8条能够免疫产生GDF15特异性抗体的抗原多肽,与VLP载体偶联后,免疫雌性BALB/c小鼠,利用杂交瘤技术制备鼠源抗人GDF15的m Ab,用间接ELISA检测m Ab腹水效价。结果:获得针对7个抗原多肽的12株稳定分泌抗人GDF15的杂交瘤细胞系,腹水m Ab效价可达1×104~1×109。结论:获得了针对不同抗原多肽的抗人GDF15的特异性m Ab,为进一步研发以GDF15为靶点的单克隆抗体抗肿瘤药物奠定了基础。     

Purification and characterization of monoclonal antibodies against the free α-subunit of human chorionic gonadotrophin

Monoclonal antibodies (Mabs) against human chorionic gonadotropin hormone (hCG) were raised by hybridoma technology using Sp2/0 myeloma cells as fusion partner. Sixty-five percent of the total culture wells exhibited hybrid growth and 8% of the total wells (13 culture wells) contained anti-hCG secreting hybrids. A positive hybrid cell line secreting antibodies against the free alpha-subunit of hCG was cloned twice by limiting dilution method and eighty four clones were obtained that secreted monoclonal antibodies anti-alpha hCG. One of these hybridoma clones (1C4) secreting monoclonal antibodies against the free alpha-subunit of hCG was selected for purification and characterization purposes. This hybridoma cell line secreted monoclonal antibodies of IgG1 subclass, which were purified by affinity chromatography on Protein A Sepharose CL-4B column with a final relative recovery of antibody activity of 75% and a purification factor of about 12. The purified preparation was analyzed by SDS-PAGE, native PAGE, and IEF. Specificity studies of this Mab revealed that it recognized specifically an epitope on the free alpha-subunits of hCG, FSH, LH, and TSH as determined by enzyme immunoassays. On the other hand, this Mab exhibited crossreactivity with other pituitary hormones either as free subunits or intact molecules as follows: alpha hCG 100%; intact hCG 1.8%; beta hCG 0.14%; alpha FSH 24.5%; intact FSH 0.8%; beta FSH 0.09%; alpha LH 20.5%; intact LH 0.9%; beta LH 0.08%; alpha TSH 50.5%; intact TSH 3.7%; beta TSH 0.07%; The affinity constant (K) of this Mab with respect to free alpha-subunit of hCG was found to be 1.5 x 10(7) I/mol as determined by the simple antibody dilution analysis method.     

庆大霉素单克隆抗体的制备及试剂盒的配制

目的建立庆大霉素直接竞争酶联免疫吸附分析方法。方法应用戊二醛法制备庆大霉素完全抗原,通过杂交瘤技术筛选分泌特异性庆大霉素抗体的杂交瘤细胞株,并建立庆大霉素竞争酶联免疫吸附分析检测方法。结果获得3株能稳定分泌庆大霉素单克隆抗体的杂交瘤细胞株,建立了庆大霉素竞争酶联免疫吸附分析检测方法,该方法操作简单具有良好的线性、特异性和精密度;庆大霉素质量浓度在1.5625～50.0000 ng/mL范围内,呈现良好的线性,r2=0.9913,50%抑制浓度为(IC50)为7.37 ng/mL,检测限(LOD)为1.54 ng/mL,该试剂盒与链霉素等8种药物无交叉反应。结论获得3株能稳定分泌庆大霉素单克隆抗体的杂交瘤细胞株,研制的庆大霉素竞争ELISA检测试剂盒具有良好的线性、特异性和精密度。     

Production of human serum albumin by sugar starvation induced promoter and rice cell culture

Human serum albumin (HSA) is the most widely used clinical serum protein. Currently, commercial HSA can only be obtained from human plasma, due to lack of commercially feasible recombinant protein expression systems. In this study, inducible expression and secretion of HSA by transformed rice suspension cell culture was established. Mature form of HSA was expressed under the control of the sucrose starvation-inducible rice α Amy3 promoter, and secretion of HSA into the culture medium was achieved by using the α Amy3 signal sequence. High concentrations of HSA were secreted into culture medium in a short time (2–4 days) by sucrose depletion after cell concentrations had reached a peak density in culture medium containing sucrose. The recombinant HSA had the same electrophoretic mobility as commercial HSA and was stable and free from apparent proteolysis in the culture medium. In a flask scale culture with repeated sucrose provision-depletion cycles, HSA was stably produced with yields up to 11.5% of total medium proteins or 15 mg/L per cycle after each sucrose provision-depletion cycle. A bubble column type bioreactor was designed for production of HSA. In the bioreactor scale culture, HSA was produced with yields up to 76.4 mg/L 4 days after sucrose depletion. HSA was purified from the culture medium to high purity by a simple purification scheme. Enrichment of HSA in culture medium simplifies downstream purification, minimizes protease degradation, and may reduce production cost. The combination of a DNA construct containing the α Amy3 promoter and signal sequence, and the use of a rice suspension cell culture can provide an effective system for the production of recombinant pharmaceutical proteins.     

Serum proteins modified by neutrophil-derived oxidants as mediators of neutrophil stimulation.

Reactive oxygen intermediates (ROI) released during inflammation may act as important mediators of neutrophil effector functions. The objective of this investigation was to evaluate the influence of ROI generation on neutrophil adhesion molecule regulation and degranulation. Induction of the neutrophil oxidative burst via Fcgamma receptor cross-linking was accompanied by up-regulation of neutrophil surface CD11b, CD35, and CD66b only in the presence of selected serum proteins, such as purified human C4, C5, or human serum albumin (HSA). Scavenging of ROI attenuated protein-dependent receptor regulations. Moreover, exogenous hydrogen peroxide was effective to increase neutrophil CD11b expression in a protein-dependent way. HSA exposed to neutrophil-derived ROI displayed signs of oxidative modification in terms of carbonyl formation. Such modified HSA transferred to resting neutrophils bound readily to the cell surface and effected receptor modulation as well as cellular spreading. In contrast, neither native HSA nor HSA protected against oxidation by the tocopherol analog Trolox exhibited agonistic properties. In conclusion, we demonstrate that neutrophil-derived ROI modify selected serum proteins, which, in turn, act as proinflammatory mediators of neutrophil stimulation.     

Studies on the mechanism of stimulation of T cells by the Mycoplasma arthritidis-derived mitogen. Role of class II IE molecules

A mitogen derived from the supernatant of broth cultures of Mycoplasma arthritidis (MAS-P) stimulates a proliferative response by normal, unprimed T cells and interleukin 2 production by some, but not all, T cell hybridomas. The response requires an IE-positive accessory cell (AC). The direct participation of IE, and not IA, in this system was confirmed by two sets of experiments. First, L cells transfected with IE, but not IA, provided effective AC function for both normal T cells and the T cell hybridoma DO-11.10. Second, we have taken a more direct approach by showing that purified IE incorporated in liposomes and used to coat glass beads can support the MAS-P response of the DO-11.10 T cell hybridoma in the absence of intact AC or other AC molecules. Although the receptor for IE-MAS-P has not been identified, we have eliminated from consideration two potential T cell recognition structures. Monoclonal antibody to the antigen-major histocompatibility complex specific receptor failed to inhibit the MAS-P response of DO-11.10 or the T cell line LBRM-33. Furthermore, the L3T4 molecule did not appear to be involved since an L3T4-negative variant of DO-11.10 responded well to the mitogen. In addition, we show that both Lyt-2-positive and L3T4-positive T cells respond to this class II-restricted stimulus. Thus, we postulate the existence of a non-T cell receptor, non-L3T4 receptor that recognizes MAS-P in association with a presumed nonpolymorphic region of IE.     

Induction of granulocyte-macrophage colony-stimulating factor by lipopolysaccharide and anti-immunoglobulin M-stimulated murine B cell lines

The present study was undertaken to elucidate whether B cell lymphoma and hybridoma cell lines can be stimulated by lipopolysaccharides (LPS) or by antibodies against immunoglobulin M (IgM) to produce granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF activity was assayed on the basophil/mast cell line PT-18 which is GM-CSF- and interleukin 3-dependent. Antibodies against murine recombinant GM-CSF were used to identify the colony-stimulating factor activity present in the supernatants of the stimulated B cell lines. When these cell lines were stimulated with LPS, two of five lymphoma and five of six hybridoma lines produced GM-CSF. Two cell lines, the B cell lymphoma M12.4.1 and the hybridoma TH2.2, were analyzed more extensively under serum-free conditions. In these two cell lines, the production of GM-CSF was dependent on the dose of LPS used and time of exposure. Antibodies against IgM stimulated the TH2.2 (IgM+) but not the M12.4.1 (IgM-) cells to produce GM-CSF. Northern blot analysis of the M12.4.1 and TH2.2 cells showed that mRNA of GM-CSF can be detected in LPS-stimulated but not in unstimulated cells. Our data show that transformed B cells can be stimulated to produce GM-CSF. The present data and previous studies on GM-CSF production by normal bone marrow-derived B cells suggest a possible participation of B cells in granulopoiesis.     

Correlation between dielectric property by dielectrophoretic levitation and growth activity of cells exposed to electric field

The purpose of this study is to develop a system analyzing cell activity by the dielectrophoresis method. Our previous studies revealed a correlation between the growth activity and dielectric property (Re[K(ω)]) of mouse hybridoma 3-2H3 cells using dielectrophoretic levitation. Furthermore, it was clarified that the differentiation activity of many stem cells could be evaluated by the Re[K(ω)] without differentiation induction. In this paper, 3-2H3 cells exposed to an alternating current (AC) electric field or a direct current (DC) electric field were cultivated, and the influence of damage by the electric field on the growth activity of the cells was examined. To evaluate the activity of the cells by measuring the Re[K(ω)], the correlation between the growth activity and the Re[K(ω)] of the cells exposed to the electric field was examined. The relations between the cell viability, growth activity, and Re[K(ω)] in the cells exposed to the AC electric field were obtained. The growth activity of the cells exposed to the AC electric field could be evaluated by the Re[K(ω)]. Furthermore, it was found that the adverse effects of the electric field on the cell viability and the growth activity were smaller in the AC electric field than the DC electric field.     

Lipid metabolism is differentially modulated by salicylic acid and heptanoyl salicylic acid during the induction of resistance in wheat against powdery mildew

Heptanoyl salicylic acid (HSA) is a salicylic acid (SA) derivative obtained by esterification of 2-OH benzoic acid with heptanoic acid. In wheat, the protection levels obtained against Blumeria graminis f. sp. tritici (Bgt) increased from 50% with SA to 95% with HSA. Using molecular, biochemical and cytological approaches, we investigated here how wheat lipid metabolism is differentially activated by SA and HSA in both infectious and non-infectious conditions, and how Bgt infectious process is altered by both inducers. First, in the absence of Bgt, continuous lipoxygenase (LOX)-encoding gene expression and corresponding activity were specifically induced by HSA. Moreover, compared to SA, HSA treatment resulted in earlier up-regulations of the phospholipase C2-encoding gene expression and it specifically affected the expression of a lipid transfer protein-encoding gene. In infectious context, both HSA and SA sprayings impaired penetration events and therefore haustorium formation, leading to less frequent fungal colonies. While this alteration only slowed down the evolution of Bgt infectious process in SA-sprayed leaves, it completely impaired the establishment of successful infectious events in HSA-sprayed leaves. In addition, HSA induced continuous increases of a LOX-encoding gene expression and of the corresponding LOX activity when compared to SA-sprayed leaves. Lipid metabolism is therefore overall highly responsive to HSA spraying and could represent effective defence mechanism triggered during the induction of resistance in wheat toward Bgt. The concepts of priming and energy costs of the defences induced by SA and HSA are also discussed.     

Tyrosine residues play an important role in heme detoxification by serum albumin

Background

Serum albumin binds avidly to heme to form heme–serum albumin complex, also called methemalbumin, and this binding is thought to protect against the potentially toxic effects of heme. However, the mechanism of detoxification has not been fully elucidated.

Methods

SDS-PAGE and Western blot were used to determine the efficiency of methemalbumin on catalyzing protein carbonylation and nitration. HPLC was used to test the formation of heme to protein cross-linked methemalbumin.

Results

The peroxidase activity of heme increased upon human serum albumin (HSA) binding. Methemalbumin showed higher efficiency in catalyzing tyrosine oxidation than free heme in the presence of H₂O₂. Methemalbumin catalyzed self-nitration and significantly promoted the nitration of tyrosine in coexistent protein, but decreased the carbonylation of coexistent protein compared with heme. The heme to protein cross-linked form of methemalbumin suggested that HSA trapped the free radical accompanied by the formation of ferryl heme. When tyrosine residues in HSA were modified by iodination, HSA lost of protection effect on protein carbonylation. The low concentration of glutathione could effectively inhibit tyrosine nitration, but had no effect on protein carbonylation.

Conclusion

HSA protects against the toxic effect of heme by transferring the free radical to tyrosine residues in HSA, therefore protecting surrounding proteins from irreversible oxidation, rather than by direct inhibiting the peroxidase activity. The increased tyrosine radicals can be reduced by endogenic antioxidants such as GSH.

General significance

This investigation indicated the important role of tyrosine residues in heme detoxification by HSA and suggested a possible novel mechanism.     

抗人呼吸道合胞病毒融合糖蛋白单克隆抗体的制备及体外鉴定

目的:获得能稳定分泌抗人呼吸道合胞病毒(human respiratory syncytial virus, RSV)融合糖蛋白(fusion glycoprotein, F)单克隆抗体(monoclonal antibody, mAb)的杂交瘤细胞株,以期用于RSV感染的早期诊断和被动免疫治疗研究。方法:通过杂交瘤技术制备可特异性识别RSV F的单抗,体外鉴定生物学特性。结果:获得了可分泌抗RSV F蛋白的杂交瘤细胞株F8,体外连续传代培养2个月,能稳定分泌抗体F8,培养上清效价为1∶1000,亲和常数(Ka)为6.8×10⁸ L/mol。F8属IgG1型抗体,可特异性识别RSV F1亚单位的AA 205-222。免疫酶法蚀斑减少中和实验证实F8具有体外中和活性及融合抑制活性。结论:获得具有中和活性的抗RSV F蛋白的单克隆抗体,为RSV感染的早期诊断及被动免疫治疗等奠定了基础。     

抗卵清蛋白单克隆抗体杂交瘤细胞株的制备

为制备分泌抗卵清蛋白的杂交瘤细胞,以高纯度的卵清蛋白抗原免疫BALB/c小鼠,取其脾脏细胞和Sp2/0骨髓瘤细胞融合,获得杂交瘤细胞,用ELISA间接法检测上清液中的抗卵清蛋白抗体效价,经3次单克隆化筛选,获得5株分泌抗卵清蛋白抗体的杂交瘤细胞株。     

抗心型脂肪酸结合蛋白单克隆抗体制备、鉴定和在侧向免疫层析方法中的应用

目的:制备抗心型脂肪酸结合蛋白(H-FABP)单克隆抗体(mAb),并建立侧向免疫层析方法检测血浆中H-FABP。方法:用H-FABP蛋白免疫纯系Balb/c小鼠,采用杂交瘤技术建立能稳定分泌抗人H-FABP的单克隆杂交瘤细胞株。常规制备腹水,纯化后得到特异性抗H-FABP单克隆抗体,进行效价、特异性、亲和力的鉴定分析,并在ELISA平台进行抗体配对,用所筛选到的抗体对初步建立了检测H-FABP的侧向免疫层析方法。结果:成功获得12株稳定分泌抗体的阳性细胞株,并筛选出能相互配对,并应用于侧向免疫层析平台的抗体3D1和5F4,检测临床样品与对照试剂比较总符合率为100%。结论:筛选能稳定分泌抗体的细胞株,配对抗体应用于侧向免疫层析检测方法中,能快速、特异、灵敏的检测出临床样品中H-FABP,为临床应用快速检测H-FABP指标提供了方法和关键材料。     

Monoclonal antibody to the region of fibronectin involved in cross-linking to human fibrin

A cross-link-containing fragment (alpha XLCNBr), derived from the alpha-polymer component of human fibrin following CNBr digestion, has been isolated and characterized. NH2-terminal sequence studies of three alpha XLCNBr derivatives, each prepared by a different method, indicate that the A alpha-chain regions comprised of residues 241-476 (CNBr VIII) and 518-584 (CNBr X) are the major constituents of the cross-linked fragments examined. Evidence for at least two additional sequences suggests that A alpha 208-235 (CNBr V) and A alpha 585-610 (CNBr XI) may have auxiliary roles in alpha-polymer formation. When alpha XLCNBr was used as an immunogen for the production of murine hybridoma cell lines, two groups of antibodies were obtained. The majority of supernatants from primary hybridoma cultures did not discriminate between fibrinogen and alpha XLCNBr and appeared to contain antibodies directed against determinants within either CNBr VIII or CNBr X [see Ehrlich, P. H., Sobel, J. H., Moustafa, Z. A., & Canfield, R. E. (1983) Biochemistry (following paper in this issue)]. Several supernatants from primary hybridoma cultures, however, did exhibit significant binding toward the alpha-polymer derivative in the absence of demonstrable immunoreactivity toward either highly purified fibrinogen or its A alpha-chain peptides, CNBr VIII and CNBr X.(ABSTRACT TRUNCATED AT 250 WORDS)     

抗细小病毒B19-VP2单克隆抗体的建立

制备抗细小病毒B19－VP2单克隆抗体,用于检测人血清中的B19抗原,辅助诊断相关疾病;也可用于制备人类细小病毒基因工程疫苗。用纯化的基因工程表达的B19－VP2蛋白免疫BALB/c小鼠,取免疫小鼠的脾细胞和小鼠骨髓瘤Sp2/0细胞融合,有限稀释法克隆细胞。ELISA及IF证明抗体特异性。克隆筛选出4株细胞,并初步建立了检测B19－VP2抗原的双抗体夹心酶联免疫吸附试验,为双抗体夹心法检测B19抗原为临床相关疾病诊断提供了检测手段。     

The effect of human serum albumin on the extended storage of human oral keratinocyte viability under mild hypothermia

Isolated oral keratinocytes in suspension provide a number of advantages for use in maxillofacial surgery, however, the poor stability of this cell preparation at physiological temperatures is an apparent barrier preventing their use. The purpose of the present study was to evaluate whether human serum albumin (HSA) could serve as an effective constituent of a storage medium to enhance human oral keratinocyte (HOK) viability under conditions of mild hypothermia. Primary human oral keratinocytes were isolated from small pieces of the non-inflamed gingival tissues obtained during the extraction of the third molars of patients. HOK were cultured on collagen type I-coated culture dishes in keratinocyte growth medium (KGM). After the trypsinization of a culture dish (passage 2 or 3), freshly isolated HOK were stored for 24, 48, and 72 h at 4 degrees C or at room temperature in KGM, saline, Dulbecco's modified Eagle's medium (DMEM), saline supplemented with 10% HSA or DMEM supplemented with 10% (v/v) HSA under one atmosphere pressure. After storage, HOK cell survival was determined by dye exclusion using trypan blue and colony-forming assay and cell cycle change was obtained by flow cytometry. Highest cell viability was obtained in saline supplemented with 10% HSA and DMEM supplemented with 10% (v/v) HSA at 4 degrees C and at room temperature. Under these conditions no significant decline in keratinocyte viability was observed for at least 48 h. The cell cycle profiles of these cells were also maintained for at least 48 h at room temperature. These observations demonstrate that HSA might be better at preserving the viability of HOK stored under hypothermic and mild hypothermic conditions up to 48 h.     

A monoclonal antibody to chicken gizzard desmin that recognizes intermediate filaments and nuclear granules in BHK21/C13 cells

One hybridoma (AC54), which produces monoclonal antibody (MAb) that recognizes both intermediate filaments (IFs) and nuclear granules in BHK21/C13 cells, and two hybridomas (AC19 and AC36) which produce MAbs that recognize IFs only, were obtained by using a crude actin preparation from chicken gizzard as an antigen. In immunoblotting, both the AC54 and AC19 MAbs reacted with the 52 kD protein (desmin) and some other proteins in gizzard and BHK21/C13 cells. Indirect immunofluorescent microscopy of BHK21/C13 cells showed that the cytoplasmic filaments stained by these MAbs were IFs based on their colchicine-induced whorl formation. The ability of AC54 MAb to recognize IFs was more limited than that of AC19 MAb. The nuclear granules recognized by AC54 MAb were in a different location than the cytoplasmic IFs and sometimes were concentrated in the nucleolus. These results indicate that AC54 MAb is an anti-desmin MAb that reacts with some desmin-related proteins; that it recognizes IFs differently than AC19 MAb, another anti-desmin MAb; and that it recognizes nuclear granules in locations where desmin or desmin-related protein has not yet been reported.     

Sulfenic acid formation in human serum albumin by hydrogen peroxide and peroxynitrite

Human serum albumin (HSA), the most abundant protein in plasma, has been proposed to have an antioxidant role. The main feature responsible for this property is its only thiol, Cys34, which comprises approximately 80% of the total free thiols in plasma and reacts preferentially with reactive oxygen and nitrogen species. Herein, we show that the thiol in HSA reacted with hydrogen peroxide with a second-order rate constant of 2.26 M(-1) s(-1) at pH 7.4 and 37 degrees C and a 1:1 stoichiometry. The formation of intermolecular disulfide dimers was not observed, suggesting that the thiol was being oxidized beyond the disulfide. With the reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl), we were able to detect the formation of sulfenic acid (HSA-SOH) from the UV-vis spectra of its adduct. The formation of sulfenic acid in Cys34 was confirmed by mass spectrometry using 5,5-dimethyl-1,3-cyclohexanedione (dimedone). Sulfenic acid was also formed from exposure of HSA to peroxynitrite, the product of the reaction between nitric oxide and superoxide radicals, in the absence or in the presence of carbon dioxide. The latter suggests that sulfenic acid can also be formed through free radical pathways since following reaction with carbon dioxide, peroxynitrite yields carbonate radical anion and nitrogen dioxide. Sulfenic acid in HSA was remarkably stable, with approximately 15% decaying after 2 h at 37 degrees C under aerobic conditions. The formation of glutathione disulfide and mixed HSA-glutathione disulfide was determined upon reaction of hydrogen peroxide-treated HSA with glutathione. Thus, HSA-SOH is proposed to serve as an intermediate in the formation of low molecular weight disulfides, which are the predominant plasma form of low molecular weight thiols, and in the formation of mixed HSA disulfides, which are present in approximately 25% of circulating HSA.