1073(le91) 91-97 '., 1991 ElsevierScience PublishersB.V. (Biomedical Division)0304-4165/91/$03.51) 030441659100053.~
Biochimit~ et Biophysica Acta. ADONIS
Antioxidant role and subcellular location of hypotaurine and taurine in human neutrophils T e r r e n c e R. G r e e n ~.2 .lack H. F e l l m a n i A l i s a L. E i c h e r ~ a n d K a t h e r i n e L. P r a t t 2 I Department of BiochemtsttT and Molecular Bmlo.v~L Oregon Ilealth S(wnces Umt'erssO'. Portlana~ OR (U.S.A) and : Chnwal Patholo,g~ St,rt'we. Veterans Adnnmstratmn .~dedt('al ('enter, Porthmd, OR I U S A.)
(Received 27 April, 1990) (Revised manuscriptreceived 3 August 19q0)
Key words: Hypotaurme"Taurine; Radical scavenger;Neurophil acti'.ation: Anlloxidant:(Human ncutrophil) The subceUular location of taurine, and its precursor, hypotaurine, within human neutrophils has been examined by nitrogen cavitation, Percoll-gradient centrifugation and HPLC analysis. Hypotatl"~ne and taurine were found to reside within the cytosolic compartment of the cell. The ratio of taurine to hypotaurine is approx 50: I. The cytosolic concentration of taurine is approx. 50 mM. The concentration of hypotaurine decreased by 80% when resting neutrophils were converted into actively respiting cells by exposure to opsonized zymosan. These r~uits prom_~ed in vitro studies on the antioxidant properties of hypotaurine. We demonstrate by EPR spectroscopy that hypo|anrine competes with 5,5'-dimethyl-l-pyrroline N-oxide) ( D M P O ) for hydroxyl radicals, and that it is the sulfinyl group which confers hydroxyl radical scavenging activity to it. Following its exposure to hydroxyl radicals, two oxidation products were isolated by HPLC, one of which has been identified as taurine. The biological roles of hypotaurine and taurine in the neutrophil are discussed with respect to their antioxidant properties and subcellular location within the cell.
Introduction Tautine, an unusual amino acid found in large quantities in the neutrophil [1-31, is a powerful scavenger of hypochlorous acid [41. It is formed by oxidation of hypotaurine [~]- Hypotaurine is a scavenger of hypochlorous acid and hydroxyl radicals [4,6,7]. Its mechanism of scavenging hydroxyl radicals has not been explored, nor has its presence in neutrophils previously been reported. We present here data on the subceUular location of hypotaurine and taurine in neutrophils, and observations on the oxidation of hypotautine accompanying activation of :esting neutrophils by opsonized zymosan. These observations are interpreted with respect to the chemical properties of hypotaurinc and
Abbreviations: EPP., ¢leetron paramagnetic resonance; DMPO. 5,5'dimethyl-l-pyrroline N-oxide" DMPO-OH. 5,5'-dimethyl-2-hydroxylpyrrolidine-l-oxyl: HBSS, Han~'*~buffered salt solution: buffer A. 0.1 M potassium phosphate. 3~, tetrahydrofuran (pH 6.8). buffer B, 0.1 M potassium phosphate. 3~ tetrahydrofuran. 40~, acetonitrile (pH 6.8). Correspondence: T.R. Green, Department of Clinical Pathology, VA Medical Center, Oregon Health Sciences University. Portland, OR 9"7207,U.S.A.
taurine as antioxidants of hypochlorous acid and hydroxyl radicals. Materials and Methods L-Cysteine, hypotautine, taurine, 7-aminobutytic acid, alanine, 5,5'-dimethyl- l-pyrroline N-oxide (DMPO), horseradish peroxidase, toluene sulfinic acid, Histopaque-1077 (leukocyte separation media), zymosan, EDTA, o-phthaldialdehyde, mercaptoethanol and Percoll were purchased from Sigma Chemicals (St. Louis, MO). Ferrous sulfate, hydrogen peroxide, sodium borate, HPLC-grade acetonitrile and methanol were obtained from J.T. Baker Chemicals (Phillipsburg, N J), Bio-Rad Bio-Sil ODS-5S reverse-phase HPLC columns (0.39 x 40 cm) were purchased from Bio-Rad Laboratoties, Richmond, CA. Tetrahydrofuran (HPLC grade) was purchased from Aldrich Chemicals (Milwaukee, Wl). H u m a n neutrophiis were isolated from venous blood by density centrifugation on ficoll-diatrizoate gradients [8]. To examine the subcellular distribution of hypotaurine and tautine, purified neutrophils were fractiona:ed by nitrogen cavitation and Percoll-gradient centtifugation [9].
Hypotaurine and taurine were quantitated by a modified HPLC method I10] which included pre-column derivatization with o-phlhaldialdehyde and anaI,jsis with a Bio-Rad Bio-Sil ODS-5S reverse-phase column. Specific conditions for operation of the HPLC column were modified as follows: flow rate, 1.5 ml per min; initial 5 rain, 8770 buffer A (0.1 M potassium phosphate, 3% fetrahydrofuran, pH 6.8) plus 137o buffer B (0.1 M potassium phosphate. 370 tetrahydrofuran, 4070 acetonitrile, pH 6.8): increased buffer B 1.47o per min over the next 5 mix and held at 2070 for an additional 5 rain; increased buffer B 9% per rain over the next 5 rain and held at 45% buffer B for an additional 10 rain: and buffer B then brought to 10070 in the next 5 rain and held for an additional 5 mix. The HPLC system included a Beckman 420 controller, two Beckman Model 110A pumps, a Beckman Model 157 fluorescence detector, and a Hewleft-Packard 3392A integrator for data recording and analysis. All hypotaurine and taurine values reported represent the average of duplicate determinations of three or more individual cell isolates as indicated in the text. Quantitation was by external standardization using authentic hypotaurine and taurine as calibrators. To examine the effect of oxidants generated by neutrophils on endogenous hypotaurine, purified cells, resuspended in Hank's buffered saline solution (HBdS) (pH 7.4) at a final concentration of approx. 1 • 107 cells per ml, were stimulated with opsonized zymosan (0.17 mg per ml cell suspension). The recovery of hypetaurine was contrasted with control experiments conducted under the same experimental conditions, excluding exposure of the cells to zymosan. Opsonized zymosan was prepared by incubating zymosan (1.7 mg per ml) at room temperature for 15 mix in 147~ serum-HBSS (pH 7.4). Unstimulated (resting) and zymosan-stimulated cells in corresponding aliquots (1 ml) were rapidly lysed by sonication on a Bronson Model 350 cell disruptor. The supernatants recovered from the crude lysa~es following protein precipitation with sulfosalicyclic acid (final concentration 3.470) were subsequently analyzed for hypotaurine and taurine content by HPLC as described above. DMPO obtained from Sigma was further purified by charcoal filtration [11]. Hydroxyl radicals were generated by Fenton chemistry by mixing ferrous sulfate (83 #M) with DMPO (71 mM) in 41 mM potassium phosphate (pH 7.8) also centaining 0.83 mM EDTA, 73 b:M hydrogen peroxide, and varying levels of hypotaurine, toluene sulfinate, or other scavengers of hydroxyl radicals. The formation of hydroxyl radicals was monitored by scanning for 5,5'-dimethyl-2-hydroxylpyrrolidineoloxyl (DMPO-OH) formation [11,12] on a Varian Model E-109 EPR spectrometer. Scan conditions, unless indicated otherwise, were as follows: microwave frequency, 9.53 kHz; power, 15 mW; modulation amplitude, 2;
modulation frequency. 100 KHz; time constant. 0.25 s; scan time. 8 min, gain 4-103. and field set, 3400 G. Results
Fig. 1 is a HPLC tracing of neutrophil lysate recovered after protein precipitation. Hypotaurine eiuted from the column at 28.57 mix; taurine had a retention time of 29.69 rain. Alanine and y-aminobutyrate, two potential sources of interference in detecting hypotaurine and taurine [10], did not interfere using the modified gradient described in Materials and Methods. Alanine eluted approx. 0.8 rain before hypotaurine, and ~,-aminobutyrate eluted approx. 0.5 min after taurine. The ratio of hypotaurine to taurine in whole cell lysates was found to be apFrox. 1 : 50. Hypotaurine and taurine were found to clearly reside within the cytosol. Less than 0,5% of the total taurine was found with the granular and plasmalemmal fractions, and hypotaurine was not detected in any fraction other than the cytosol. Table I summarizes the distribution of hypotaurine and taurine among subcellular fractions. Hypotaurine levels varied depending upon whether cells were assessed before or after exposure to stimulants eliciting an oxidative burst response. The hypotaurine and taurine content of resting (unexposed) neutrophils was 0.37 (range 0.33-0.40, n = 3) and 21.0 (range 19.4-21.9, n = 3) nmol per 106 cells, respectively. Assummg a uniform distribution in the cytosol fraction, a cell volume of 450 fl [13], and taking into account the total nmol recovered and cell count used in these experiments, we calculate the approximate concentrations of hypotaurine and taurine in the cell be 0.8 and 48 raM, respectively. After 30 min exposure to opsonized zymosan we obtained values of 0.07 (range
~ - - ~
....... ;b- ....... ,[ .... z'n
" ~
"
-~b ..... f5
Fime (rain) Fig. l. HPLC tracing of o-pthaldia!dchydc-trcatcd ncutrophil lysate. 50 pl of protein-free lysate (see Materials and Methods) diluted
t0-fold in distilled water was mixed with 50 pl of o-pthaldialdehyde reagent (10). allowed to react approx, t rain at room temperature. then diluted to 1 ml in 0.l M phosphate buffer (pH 6.8); aliquot injected. 250 ttl; P.uoresccnce settings, excitation at 360 nm and emission at 450 nm; arrow~mark the elutioo peaks for hypotaurine (HT) and taurine(T). Developmentof the mobilepha,~ and quantit~tion wereas in Materials and Me:hods.
TABLE I Suhcellular Iocatmn of hvpotourme and tuurme m re~ttng neutrop/td~ "
Sub'=ellularfraction
Hypotaurme
Taurtae
(nmol)
(nmol)
cytosol
Igl ± 5.6 (n = 3,
8568 + 94 (n = 3)
p!asmalemma
n.d.
23.6 + 2 ( n = 3)
spectfic granules
n.d.
148 +_1 6 (n = 3)
azurophilic granules
n.d
0.,~ +_08 (,, ~ 3)
a ~,alues represent the average + 1 S.D. of three separale ~.eterminalions on each subcellular fraction, n.d. = none detccte]. Purified neutrophi|s were fractionated by nitrogen cavitation ~nd Percollgradient centrifugation as indicated in Materials and 'Method:,
0-0.156, n = 4) nmol %r hypotaurine per 10 ~ cells and 18.7 (range 13.8-|9.9, n = 4) nmol for tauriuc per I0 ~ cells. These d e t e r m i n a t i o n s represent the average and range of values o b t a i n e d from duplicate assays conducted on multiple isolates ( n ) from the same source of cells used in the resting cell experiments. Hence, there was roughly an 80% decrease in hypotaurine content associated with the conversion of resting neutrophils into actively respiring cells, but no apparent change in taurine levels under the same experimental conditions. Since no information is presently available regarding the specific action of neutrophil oxidants on hypotaurine, we detet,nined it would not be feasible to ascertain the precise mechanism(,) by which hypotaurine was metabolized without additional information on Itow hypotaurine behaves when exposed to oxidants u n d e r more controlled conditions. The oxidizing action of hypochlorous acid on hypotaurine has been extensively studied [4]. Little is known regarding the action of hydroxyl radicals on hypotaurine. Hence we focused on the interaction of h y p o t a u r i n e with hydroxyl radicals under defined conditions in anticipation that this inform a t i o n might yield useful ways of d i s c r i m i n a t i n g between hydroxyl radical mediated, as opposed to hypochlorous acid mediated, oxidation of hypotaurine in whole cell isolates. These studies were prefaced on the a s s u m p t i o n that hypochlorous acid and hydroxyl radicals are the two most likely oxidants accounting for turnover of hypotaurine inside the neutrophil (see Discussion). Cell-free reaction mixtures m a d e up of ferrous sulfate a n d hydrogen peroxide and including D M P O were prepared to allow for more controlled generation and assessment of hydroxyl radical formation (see Materials and Methods). Fig. 2 d e m o n s t r a t e s the characteristic 1 : 2 : 2 : 1 E P R resonances and splitt:mg constants (A s = A n = 15 G ) of D M P O - O H fcrmcd in the absence
(Fig. 2a) and presence of 10 m M (Fig. 2b) and 41 mM (Fig. 2c) hypotaurine. Increasing only the concentration of hypotaurine in the reaction mixtures results in a marked decrease in D M P O - O H trapped. Identical inhibition data and spectral scans were obtained by substituting toluene sulfinate for hypotaurine in the reaction mixt'-,re>. Fig. 3 c o m p a r e s the scavenging activity of hypotaurine wiln that obtained by toluene sulfinate. Since toluene sulfmate dnd h y p o t a u r i n e are structurally completely different from one another except for the sulfinyl group, the identical hydroxyl radical scavenging activitie,- on a millimolar basis [cf. Fig. 3) strongly suggested that the sulfinyl group was responsible for hydroxyl radical scavenging activity. Although the sulfinyl group appeared to be the site of hydroxyl radical attack, we were pt~zzled by the absence of any evidence for formation of t h i y I - D M P O adducts expected for the reaction of thiyl radical intermediates wi,h DMPO. Saez et al. [14] have previously shown that t h i y I - D M P O adducts form rapidly and are easily delectable by E P R following exposure of cysteine to, hydrogen peroxide. H a r m a n et al. [15] have demonstrated with horseraddish peroxidase and hydrogen per-
J J
S
+
lOG
Fig. 2. Scavenging of hydroxyl radicals by hypotaurln¢. (a) ferrous sulfate (83 taM), [)MOO (71 mM) in 41 mM potassium phosphate (pH 7.8), 0.83 mM EDTA and 13g/~M hydrogen peroxide; (b) same as (a) + 10 mM hypo,aurin¢; and (c) same as (a) +41 mM hypotaurin¢. Scans wcrc made 8 rain after initiation of the reaction on a Varian E-109 ~criesEPR spectrometer. Scan conditions: microwave frequenc), 9.53 kHz; power, 15 roW; modulation amplitude. 2" m,.'dulation frequency. 100 kHz: time constant, 0.25 s; s'=an time, 8 rain; gain, 4.10~; and field ~t, 3400 (3.
,oo
80"
~
./it
60
40
,.x ° ~ ',' °
*~~~~"
........... 4'0 60 80 I00 Concentrotion(mM) Fig. 3. Scavengmg capacity of hypotaurine and toluene sulfinate in ~'o
competing for hydloxyl radicals against DMPO. Percent inhibition represents the percent decrease in DMPO-OH formation caused by varying the concentration of hypotaurine (0) and toluene sulfinate (X), while holding all other reaction conditions constant. Hydroxyl radicals were generated and monitored by EPR as in Fig. 2a but with inclusion of hypotaurine or toluene sulfinate at varying concentrations as indicated in the abscissa. Pc.cent inhibition of DMPO-OH formation was calculated as the decrease in height of the main resonance signal compared to signals obtained in the absence of hypotaurine and toluene sulfinate. oxide the formation of t h i y I - D M P O adducts o | cysteine and cysteine sulfinic acid. Under similar conditions to those used by H a r m a n et al. we confirmed formation of thiyl adducts of D M P O with cysteine, but not hypotaurine, and a thiyl-like a d d u c t of D M P O for toluene sulfinate. EPR scans were conducted as in Fig. 2 for D M P O - O H formation. Freshly prepared solutions of 41 m M cysteine, hypotaurine and toluene sulfinate in a final concentration of 41 m M p h o s p h a t e buffer (pH 7.8) supplemented with 83 ttM E D T A and 3.5 mg per ml horseraddish peroxidase were individually scanned for DMPO-thiyl adduct formation approx. 15 rain after the addition of hydrogen peroxide (final concentration 3.6 mM). Resonance signals characteristic of t h i y I - D M P O adducts were easily detected at gain settings of 4 . 1 0 3 for cysteine and toluene sulfinate. H y p o t a u r i n e was not readily oxidized under the above experimental conditions. A very weak resonance spectrum resembling D M P O - O H , but lacking any a p p a r e n t trace of thiyl a d d u c t was observed at a higher gain setting of 1 • 10 4. For the thiyl adduct of cysteine we obtained splitting constants of A N = 15.3 G and A , = 17.8 G. which are in reasonably good agreement with those reported by H a r m a n et al. [15] and Saez et al. [14]. For toluene sulfinate we observed splitting constants of Ar~ = 13.8 G and A H = 16 G with the resonance spectrum appearing qualitatively quite similar to that of the thiyl a d d u c t of cysteine. Omission of the thiyl subst[ates yielded fiat baselines. Fig. 4 shows typical spectral scans obtained following 15 min exposure of the reaction mixtures to horseraddish peroxidase and hydrogen peroxide. These experiments indicated that we were capable of detecting thiyl adducts of D M P O . Yet with hydroxyl
radicals serving as oxidants, we saw no evidence of t h i y I - D M P O a d d u c t formation. The absence of any spectra resembling t h i y l - D M P O adducts with hydroxyl radicals serving as oxidants of h y p o t a u r i n e and toluene sulfinate suggests that the sulfinyl moiety does not last as a free-radical intermediate for sufficient time in the presence of hydroxyl radicals to a c c u m u l a t e and condense with D M P O to yield a thiyl adduct. As such, we concluded that the sulfinyl radical i n t e r m e d i a t e s must rapidly convert, in the presence of hydroxyl radicals, to oxidation products beyond t r a p p i n g by D M P O . W e therefore sought to assess whether o x i d a t i o n p r o d u c t s of hypotaurine might be detectable by direct c h r o m a t o graphy of the Fenton reaction mixtures where hydroxyl radical scavenging had previously been observed, but where, by EPR analysis, no t h i y I - D M P O a d d u c t s appeared to accumulate. Experiments were c o n d u c t e d by exposing stock 20 m M ~olutions of freshly prepared hypotaurine m a d e up in 41 m M p h o s p h a t e buffer (pH 7.8) also c o n t a i n i n g 83 m M E D T A to ferrous sulfate, hydrogen peroxide, and mixtures of ferrous sulfate and hydrogen peroxide. F o l l o w i n g a 15 rain i n c u b a t i o n at
lOG
C
lOG
Fig. 4. EPR spectralscansof DMPO products fcrmed by enzymatic peroaidation of cysteine, toluene sulfinate and hypotaurine. Thiyl substrate (final concentration 41 raM) in 41 mM phosphate buffer (pH 7.8), also supplemented with 0.83 mM EDTA and 71 mM DMPO was mixed with hydrogen peroxide (final concentration 3.9 raM) and horseraddish peroxidasc (final concentration 0.5 mg per ml). Scans were made approx. 8 rain after the addition of horseraddish peroxidase and hydrogen peroxide with EPR settings as in Fig. 2 except for the gain which was increased to I. 104. Spectral scans shown are with: a, cysteine; b, toluene sutfinate; c, hypotaurine; and d. control scan conducted in the absence of thiyl subs~rate.
95 room temperature, 10 to 20 #1 aliquots of the reaction mixtures were derivitized with o-phthaldialdehyde and immediately analyzed. By analysis of the relative differences in peak recoveries and retention times among the samples tested, two resolvable oxidation products of hypotaurine were detected. One of the reaction products was identified as taurine. The more polar product (suggested by its shorter retention time on the reverse-phase column) has not yet been identified. Chromatographic conditions were as follows for observation of these oxidation products: column starting buffer, 5% buffer B in buffer A held constant for the first 5 rain after injection of the sample at a flow rate of 0.9 ml per re!n:
I
lYr it
:~T
i "
'
I
.
!I
T,me (re,n)
o
T,me (m:n)
l i I, .T :i
I) iscu.~ion
I; '!
i
°
tI,.T !i
:i
i;
!
thereafter, the concentration of buffer B was increased by 1.4c~c per rain linearly to 100% buffer B; the column was re-equilibrated in starting mobile-phase buffer to 15 rain after holding at 100% B for 10 min. Fig. 5a shows the effect of incubating hypotaurine in phosphate-EDTA buffer free of ferrous sulfate and hydrogen peroxide compared to incubations conducted in the presence of hydrogen peroxide (Fig. 5b), ferrous sulfate (Fig. 5c), and a mixture of ferrous sulfate and hydrogen peroxide (Fig. 5d). liypotaurine was recovered in these HPLC separations at a retention time of approx. 40.3 rain. Taurine (present as a contaminant in the starting stock solution of hypotaurine at an identical concentration of al~prox. 0.9%) eluted with a retention time of 41.2 min. Upon exposing hypotaurine to ferrous sulfate the taurine level rose to 3.7%. lit the presence of hydrogen peroxide taurine rose to 2.2%. With hypotaurine exposed to ferrous sulfate and hydrogen peroxide, approx. 5.7% of the hypotaurine was converted into taurine, and an additional 7.5% was detected in the form of a polar compound eluting with a short retention time of approx. 6.4 rain (of. Fig. 5d).
li,
T~me[rain) Ttme (.T:inI Fig. 5. Detection of oxidauon products of hypotaurine following exposure to hydroxyl radicals. Hypotaurine (20 raM) was allowcd to react with hydrogen peroxide and ferrous sulfate for approx. 15 min as in Fig. 2, but wtth the omission of DMPO. Aliquots were then dehvitized with o-pthaldialdehyde as in Fig. I and subjected to HPLC separation as in Materials and Methods. The mobile phase was modified as follows: startin 8 buffer, 5% buffer B in buffer A held
constant for 5 win after injection; thereafter, increase buffer B by 1.4% per ntin at a fixed flow rate of 0.9 ml per rain to a final concentration of buffer B of 100% I = HPLC injection started; HT = hypotaurine; T = taurine; and U = unidentified oxidationproduct (a) Hypotaurine without exposure to hydrogen peroxide and ferrous sulfate. (b) Hypotaurine+ hydrogen peroxide alone. (c) Hypotaurine + ferrous sulfate alone. (d) Hypotaurine+ ferrous sulfate+ hydrogen peroxide (completereaction mixture).
The detection of hypotaurine in the neutrophil has not heretofore been reported. Neutrophils are known to elicit a respiratory burst response to extracellular stimuli culminating in the extracellular release of several oxidant species including superoxide, hydrogen peroxide, hypochlorous acid and, possibly, hydroxyl radicals ]16l~l. Wasil eta!. [!9] and Gfisham el al [7~]have presented data which indicates that taurine acts as ae antioxidant in quenching the oxidizing activity of hypochlorous acid. in the present study the concentration of intracellular hypotaurine was observed to decrease approx. 80% concomitant with conversion of resting neutrophils into actively respiring cells with opsonized zymosan. This suggests that the biological role of hypotaurine, like that of taurine, is linked to the release of oxidants accompanying activation of resting neutrophils. Taurine chloramine formation would be the expected endproduct of hypotautine oxidation [4]. Since little is understood regarding the mechanism of hydroxyl radical mediated oxidation of hypotaurine0 we chose to investigate this latter reaction by exposure of hypotaurine to hydroxyl radicals generated via Fenton chemistry to gain a clearer insight as to the fate of hydroxyl radical mediated oxidation of hypotaurine. As evident in the experiments summarized in Fig. 5, hypotaurine was converted to taurine under conditions in which hydroxyl radical generation would be expected to occur via a Fenton reaction. Hence. laurine formation was seen upon exposure of hypotaurine to either ferrous sulfate or a combination of ferrous sulfate and hydrogen peroxide. The formation of taurine by hydroxyl
96 radical mediated oxidation of hypotaurine most likely occurs by the formation of a thiyl radical intermediate tot) unstable to be trapped by DMPO. Since oxidation of the sulfur moeity by hydroxyl radict, l would be expected 1o lead to formation of thiyl radical, and because we have previously demonstrated by mass spectral analysis formation of disulfon_e intermediates of hypotaurine under these experimental conditions [6], we believe it is reasonable to conclude that the rate of thiyl radical dimerization exceeds that of thiyl-DMPO formation. This would explain why thiyl-DMPO adducts of hypotaurine were not seen when hypotaurine was oxidized by Fenton chemistry in the presence of DMPO (cf. Fig. 2). On theoretical grounds the disulfone intermediate would be expected to hydrolyze to hypotaurine and taurine. Thus hydroxyl radical mediated oxidation of hypotaurine leading to taurin*, formation likely progresses through the following series of reactions: hypotaurine + hydroxyl radical ~ sulfinyl radical
hydroxyl radicals, and since hypochlorous acid is a well-recognized product of respiring neutrophils, it is clear that measurements of hypotaurine turnover accompanying cell activation and expression of a respiratory burst response cannot serve as evidence that neutrophils produce hydroxyl radicals. However, it is possible that some oxidation products of hypotaurine, such as the disulfone intermediate, may be sufficiently specific products of hydroxyl radical mediated oxidation as to reflect its formation in neutrophils. In this respect, further work in charac*erizing specific h?droxyl radical mediated oxidation products of hypotaurine, and a search for their presence in respiring neutrophils. appears warranted. Studies along these lines should lead 1o a clearer understanding of the antioxidant role of hypotaurine and taurine in addition to the nature of the oxidants reaching the intracellular spaces of the neutrophil where these two unusual amino acids reside. Acknowledgements
2 sulfinyl radicals ---, disulfone disulfone + hydroxide ---, hypotaurine + taurine At the present time, insufficient product has been isolated from the HPLC column to ascertain whether or not the polar oxidation product shown in Fig. 5d is the disulfone intermediate. Hence, we do not know whether the polar peak is the a-disulfone intermediate, or a third, yet unidentified, oxidation product of hypotaurine. Further work will be required in determining its chemical structure before a search for hydroxyl radical mediated oxidation products of hypotaurine in respiring neutrophil suspensions can be conducted. Since neutrophils direct their oxidative activity extracellularly in killing bacteria, whereas hypotaurine and taurine clearly reside within the cytosol (cf. Table I), the oxidation of hypotaurine and taurine residing in the cytosolic compartment following cell stimulation may at first appear enigmatic. However, Albrich et al. [20] have shown that hypochlorous acid bleaches carotene embedded in bacterial cell walls. They interpreted these results as evidence that cell membranes pose no significant barrier to hypochlorous acid. Ohno et al. [21] have obtained cytochemical evidence that hydrogen peroxide formed by respiring neutrophils crosses the neutrophil membrane and enters the cytoplasmic space. The presence of hydrogen peroxide and transition metals in the cytoplasmic compartment may also allow for Fenton-type reactions to occur within the cytosol. Hence, on chemical grounds it appears that the neutrophil cytosol fraction is not free from oxidative assault. Since the chemistry of hypotaurine is such that it is readily oxidized by hypochlorous acid in addition to
This work was supported by the Medical Research Foundation of Oregon and the Veterans Administration. References
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Biochimit~ et Biophysica Acta. ADONIS
Antioxidant role and subcellular location of hypotaurine and taurine in human neutrophils T e r r e n c e R. G r e e n ~.2 .lack H. F e l l m a n i A l i s a L. E i c h e r ~ a n d K a t h e r i n e L. P r a t t 2 I Department of BiochemtsttT and Molecular Bmlo.v~L Oregon Ilealth S(wnces Umt'erssO'. Portlana~ OR (U.S.A) and : Chnwal Patholo,g~ St,rt'we. Veterans Adnnmstratmn .~dedt('al ('enter, Porthmd, OR I U S A.)
(Received 27 April, 1990) (Revised manuscriptreceived 3 August 19q0)
Key words: Hypotaurme"Taurine; Radical scavenger;Neurophil acti'.ation: Anlloxidant:(Human ncutrophil) The subceUular location of taurine, and its precursor, hypotaurine, within human neutrophils has been examined by nitrogen cavitation, Percoll-gradient centrifugation and HPLC analysis. Hypotatl"~ne and taurine were found to reside within the cytosolic compartment of the cell. The ratio of taurine to hypotaurine is approx 50: I. The cytosolic concentration of taurine is approx. 50 mM. The concentration of hypotaurine decreased by 80% when resting neutrophils were converted into actively respiting cells by exposure to opsonized zymosan. These r~uits prom_~ed in vitro studies on the antioxidant properties of hypotaurine. We demonstrate by EPR spectroscopy that hypo|anrine competes with 5,5'-dimethyl-l-pyrroline N-oxide) ( D M P O ) for hydroxyl radicals, and that it is the sulfinyl group which confers hydroxyl radical scavenging activity to it. Following its exposure to hydroxyl radicals, two oxidation products were isolated by HPLC, one of which has been identified as taurine. The biological roles of hypotaurine and taurine in the neutrophil are discussed with respect to their antioxidant properties and subcellular location within the cell.
Introduction Tautine, an unusual amino acid found in large quantities in the neutrophil [1-31, is a powerful scavenger of hypochlorous acid [41. It is formed by oxidation of hypotaurine [~]- Hypotaurine is a scavenger of hypochlorous acid and hydroxyl radicals [4,6,7]. Its mechanism of scavenging hydroxyl radicals has not been explored, nor has its presence in neutrophils previously been reported. We present here data on the subceUular location of hypotaurine and taurine in neutrophils, and observations on the oxidation of hypotautine accompanying activation of :esting neutrophils by opsonized zymosan. These observations are interpreted with respect to the chemical properties of hypotaurinc and
Abbreviations: EPP., ¢leetron paramagnetic resonance; DMPO. 5,5'dimethyl-l-pyrroline N-oxide" DMPO-OH. 5,5'-dimethyl-2-hydroxylpyrrolidine-l-oxyl: HBSS, Han~'*~buffered salt solution: buffer A. 0.1 M potassium phosphate. 3~, tetrahydrofuran (pH 6.8). buffer B, 0.1 M potassium phosphate. 3~ tetrahydrofuran. 40~, acetonitrile (pH 6.8). Correspondence: T.R. Green, Department of Clinical Pathology, VA Medical Center, Oregon Health Sciences University. Portland, OR 9"7207,U.S.A.
taurine as antioxidants of hypochlorous acid and hydroxyl radicals. Materials and Methods L-Cysteine, hypotautine, taurine, 7-aminobutytic acid, alanine, 5,5'-dimethyl- l-pyrroline N-oxide (DMPO), horseradish peroxidase, toluene sulfinic acid, Histopaque-1077 (leukocyte separation media), zymosan, EDTA, o-phthaldialdehyde, mercaptoethanol and Percoll were purchased from Sigma Chemicals (St. Louis, MO). Ferrous sulfate, hydrogen peroxide, sodium borate, HPLC-grade acetonitrile and methanol were obtained from J.T. Baker Chemicals (Phillipsburg, N J), Bio-Rad Bio-Sil ODS-5S reverse-phase HPLC columns (0.39 x 40 cm) were purchased from Bio-Rad Laboratoties, Richmond, CA. Tetrahydrofuran (HPLC grade) was purchased from Aldrich Chemicals (Milwaukee, Wl). H u m a n neutrophiis were isolated from venous blood by density centrifugation on ficoll-diatrizoate gradients [8]. To examine the subcellular distribution of hypotaurine and tautine, purified neutrophils were fractiona:ed by nitrogen cavitation and Percoll-gradient centtifugation [9].
Hypotaurine and taurine were quantitated by a modified HPLC method I10] which included pre-column derivatization with o-phlhaldialdehyde and anaI,jsis with a Bio-Rad Bio-Sil ODS-5S reverse-phase column. Specific conditions for operation of the HPLC column were modified as follows: flow rate, 1.5 ml per min; initial 5 rain, 8770 buffer A (0.1 M potassium phosphate, 3% fetrahydrofuran, pH 6.8) plus 137o buffer B (0.1 M potassium phosphate. 370 tetrahydrofuran, 4070 acetonitrile, pH 6.8): increased buffer B 1.47o per min over the next 5 mix and held at 2070 for an additional 5 rain; increased buffer B 9% per rain over the next 5 rain and held at 45% buffer B for an additional 10 rain: and buffer B then brought to 10070 in the next 5 rain and held for an additional 5 mix. The HPLC system included a Beckman 420 controller, two Beckman Model 110A pumps, a Beckman Model 157 fluorescence detector, and a Hewleft-Packard 3392A integrator for data recording and analysis. All hypotaurine and taurine values reported represent the average of duplicate determinations of three or more individual cell isolates as indicated in the text. Quantitation was by external standardization using authentic hypotaurine and taurine as calibrators. To examine the effect of oxidants generated by neutrophils on endogenous hypotaurine, purified cells, resuspended in Hank's buffered saline solution (HBdS) (pH 7.4) at a final concentration of approx. 1 • 107 cells per ml, were stimulated with opsonized zymosan (0.17 mg per ml cell suspension). The recovery of hypetaurine was contrasted with control experiments conducted under the same experimental conditions, excluding exposure of the cells to zymosan. Opsonized zymosan was prepared by incubating zymosan (1.7 mg per ml) at room temperature for 15 mix in 147~ serum-HBSS (pH 7.4). Unstimulated (resting) and zymosan-stimulated cells in corresponding aliquots (1 ml) were rapidly lysed by sonication on a Bronson Model 350 cell disruptor. The supernatants recovered from the crude lysa~es following protein precipitation with sulfosalicyclic acid (final concentration 3.470) were subsequently analyzed for hypotaurine and taurine content by HPLC as described above. DMPO obtained from Sigma was further purified by charcoal filtration [11]. Hydroxyl radicals were generated by Fenton chemistry by mixing ferrous sulfate (83 #M) with DMPO (71 mM) in 41 mM potassium phosphate (pH 7.8) also centaining 0.83 mM EDTA, 73 b:M hydrogen peroxide, and varying levels of hypotaurine, toluene sulfinate, or other scavengers of hydroxyl radicals. The formation of hydroxyl radicals was monitored by scanning for 5,5'-dimethyl-2-hydroxylpyrrolidineoloxyl (DMPO-OH) formation [11,12] on a Varian Model E-109 EPR spectrometer. Scan conditions, unless indicated otherwise, were as follows: microwave frequency, 9.53 kHz; power, 15 mW; modulation amplitude, 2;
modulation frequency. 100 KHz; time constant. 0.25 s; scan time. 8 min, gain 4-103. and field set, 3400 G. Results
Fig. 1 is a HPLC tracing of neutrophil lysate recovered after protein precipitation. Hypotaurine eiuted from the column at 28.57 mix; taurine had a retention time of 29.69 rain. Alanine and y-aminobutyrate, two potential sources of interference in detecting hypotaurine and taurine [10], did not interfere using the modified gradient described in Materials and Methods. Alanine eluted approx. 0.8 rain before hypotaurine, and ~,-aminobutyrate eluted approx. 0.5 min after taurine. The ratio of hypotaurine to taurine in whole cell lysates was found to be apFrox. 1 : 50. Hypotaurine and taurine were found to clearly reside within the cytosol. Less than 0,5% of the total taurine was found with the granular and plasmalemmal fractions, and hypotaurine was not detected in any fraction other than the cytosol. Table I summarizes the distribution of hypotaurine and taurine among subcellular fractions. Hypotaurine levels varied depending upon whether cells were assessed before or after exposure to stimulants eliciting an oxidative burst response. The hypotaurine and taurine content of resting (unexposed) neutrophils was 0.37 (range 0.33-0.40, n = 3) and 21.0 (range 19.4-21.9, n = 3) nmol per 106 cells, respectively. Assummg a uniform distribution in the cytosol fraction, a cell volume of 450 fl [13], and taking into account the total nmol recovered and cell count used in these experiments, we calculate the approximate concentrations of hypotaurine and taurine in the cell be 0.8 and 48 raM, respectively. After 30 min exposure to opsonized zymosan we obtained values of 0.07 (range
~ - - ~
....... ;b- ....... ,[ .... z'n
" ~
"
-~b ..... f5
Fime (rain) Fig. l. HPLC tracing of o-pthaldia!dchydc-trcatcd ncutrophil lysate. 50 pl of protein-free lysate (see Materials and Methods) diluted
t0-fold in distilled water was mixed with 50 pl of o-pthaldialdehyde reagent (10). allowed to react approx, t rain at room temperature. then diluted to 1 ml in 0.l M phosphate buffer (pH 6.8); aliquot injected. 250 ttl; P.uoresccnce settings, excitation at 360 nm and emission at 450 nm; arrow~mark the elutioo peaks for hypotaurine (HT) and taurine(T). Developmentof the mobilepha,~ and quantit~tion wereas in Materials and Me:hods.
TABLE I Suhcellular Iocatmn of hvpotourme and tuurme m re~ttng neutrop/td~ "
Sub'=ellularfraction
Hypotaurme
Taurtae
(nmol)
(nmol)
cytosol
Igl ± 5.6 (n = 3,
8568 + 94 (n = 3)
p!asmalemma
n.d.
23.6 + 2 ( n = 3)
spectfic granules
n.d.
148 +_1 6 (n = 3)
azurophilic granules
n.d
0.,~ +_08 (,, ~ 3)
a ~,alues represent the average + 1 S.D. of three separale ~.eterminalions on each subcellular fraction, n.d. = none detccte]. Purified neutrophi|s were fractionated by nitrogen cavitation ~nd Percollgradient centrifugation as indicated in Materials and 'Method:,
0-0.156, n = 4) nmol %r hypotaurine per 10 ~ cells and 18.7 (range 13.8-|9.9, n = 4) nmol for tauriuc per I0 ~ cells. These d e t e r m i n a t i o n s represent the average and range of values o b t a i n e d from duplicate assays conducted on multiple isolates ( n ) from the same source of cells used in the resting cell experiments. Hence, there was roughly an 80% decrease in hypotaurine content associated with the conversion of resting neutrophils into actively respiring cells, but no apparent change in taurine levels under the same experimental conditions. Since no information is presently available regarding the specific action of neutrophil oxidants on hypotaurine, we detet,nined it would not be feasible to ascertain the precise mechanism(,) by which hypotaurine was metabolized without additional information on Itow hypotaurine behaves when exposed to oxidants u n d e r more controlled conditions. The oxidizing action of hypochlorous acid on hypotaurine has been extensively studied [4]. Little is known regarding the action of hydroxyl radicals on hypotaurine. Hence we focused on the interaction of h y p o t a u r i n e with hydroxyl radicals under defined conditions in anticipation that this inform a t i o n might yield useful ways of d i s c r i m i n a t i n g between hydroxyl radical mediated, as opposed to hypochlorous acid mediated, oxidation of hypotaurine in whole cell isolates. These studies were prefaced on the a s s u m p t i o n that hypochlorous acid and hydroxyl radicals are the two most likely oxidants accounting for turnover of hypotaurine inside the neutrophil (see Discussion). Cell-free reaction mixtures m a d e up of ferrous sulfate a n d hydrogen peroxide and including D M P O were prepared to allow for more controlled generation and assessment of hydroxyl radical formation (see Materials and Methods). Fig. 2 d e m o n s t r a t e s the characteristic 1 : 2 : 2 : 1 E P R resonances and splitt:mg constants (A s = A n = 15 G ) of D M P O - O H fcrmcd in the absence
(Fig. 2a) and presence of 10 m M (Fig. 2b) and 41 mM (Fig. 2c) hypotaurine. Increasing only the concentration of hypotaurine in the reaction mixtures results in a marked decrease in D M P O - O H trapped. Identical inhibition data and spectral scans were obtained by substituting toluene sulfinate for hypotaurine in the reaction mixt'-,re>. Fig. 3 c o m p a r e s the scavenging activity of hypotaurine wiln that obtained by toluene sulfinate. Since toluene sulfmate dnd h y p o t a u r i n e are structurally completely different from one another except for the sulfinyl group, the identical hydroxyl radical scavenging activitie,- on a millimolar basis [cf. Fig. 3) strongly suggested that the sulfinyl group was responsible for hydroxyl radical scavenging activity. Although the sulfinyl group appeared to be the site of hydroxyl radical attack, we were pt~zzled by the absence of any evidence for formation of t h i y I - D M P O adducts expected for the reaction of thiyl radical intermediates wi,h DMPO. Saez et al. [14] have previously shown that t h i y I - D M P O adducts form rapidly and are easily delectable by E P R following exposure of cysteine to, hydrogen peroxide. H a r m a n et al. [15] have demonstrated with horseraddish peroxidase and hydrogen per-
J J
S
+
lOG
Fig. 2. Scavenging of hydroxyl radicals by hypotaurln¢. (a) ferrous sulfate (83 taM), [)MOO (71 mM) in 41 mM potassium phosphate (pH 7.8), 0.83 mM EDTA and 13g/~M hydrogen peroxide; (b) same as (a) + 10 mM hypo,aurin¢; and (c) same as (a) +41 mM hypotaurin¢. Scans wcrc made 8 rain after initiation of the reaction on a Varian E-109 ~criesEPR spectrometer. Scan conditions: microwave frequenc), 9.53 kHz; power, 15 roW; modulation amplitude. 2" m,.'dulation frequency. 100 kHz: time constant, 0.25 s; s'=an time, 8 rain; gain, 4.10~; and field ~t, 3400 (3.
,oo
80"
~
./it
60
40
,.x ° ~ ',' °
*~~~~"
........... 4'0 60 80 I00 Concentrotion(mM) Fig. 3. Scavengmg capacity of hypotaurine and toluene sulfinate in ~'o
competing for hydloxyl radicals against DMPO. Percent inhibition represents the percent decrease in DMPO-OH formation caused by varying the concentration of hypotaurine (0) and toluene sulfinate (X), while holding all other reaction conditions constant. Hydroxyl radicals were generated and monitored by EPR as in Fig. 2a but with inclusion of hypotaurine or toluene sulfinate at varying concentrations as indicated in the abscissa. Pc.cent inhibition of DMPO-OH formation was calculated as the decrease in height of the main resonance signal compared to signals obtained in the absence of hypotaurine and toluene sulfinate. oxide the formation of t h i y I - D M P O adducts o | cysteine and cysteine sulfinic acid. Under similar conditions to those used by H a r m a n et al. we confirmed formation of thiyl adducts of D M P O with cysteine, but not hypotaurine, and a thiyl-like a d d u c t of D M P O for toluene sulfinate. EPR scans were conducted as in Fig. 2 for D M P O - O H formation. Freshly prepared solutions of 41 m M cysteine, hypotaurine and toluene sulfinate in a final concentration of 41 m M p h o s p h a t e buffer (pH 7.8) supplemented with 83 ttM E D T A and 3.5 mg per ml horseraddish peroxidase were individually scanned for DMPO-thiyl adduct formation approx. 15 rain after the addition of hydrogen peroxide (final concentration 3.6 mM). Resonance signals characteristic of t h i y I - D M P O adducts were easily detected at gain settings of 4 . 1 0 3 for cysteine and toluene sulfinate. H y p o t a u r i n e was not readily oxidized under the above experimental conditions. A very weak resonance spectrum resembling D M P O - O H , but lacking any a p p a r e n t trace of thiyl a d d u c t was observed at a higher gain setting of 1 • 10 4. For the thiyl adduct of cysteine we obtained splitting constants of A N = 15.3 G and A , = 17.8 G. which are in reasonably good agreement with those reported by H a r m a n et al. [15] and Saez et al. [14]. For toluene sulfinate we observed splitting constants of Ar~ = 13.8 G and A H = 16 G with the resonance spectrum appearing qualitatively quite similar to that of the thiyl a d d u c t of cysteine. Omission of the thiyl subst[ates yielded fiat baselines. Fig. 4 shows typical spectral scans obtained following 15 min exposure of the reaction mixtures to horseraddish peroxidase and hydrogen peroxide. These experiments indicated that we were capable of detecting thiyl adducts of D M P O . Yet with hydroxyl
radicals serving as oxidants, we saw no evidence of t h i y I - D M P O a d d u c t formation. The absence of any spectra resembling t h i y l - D M P O adducts with hydroxyl radicals serving as oxidants of h y p o t a u r i n e and toluene sulfinate suggests that the sulfinyl moiety does not last as a free-radical intermediate for sufficient time in the presence of hydroxyl radicals to a c c u m u l a t e and condense with D M P O to yield a thiyl adduct. As such, we concluded that the sulfinyl radical i n t e r m e d i a t e s must rapidly convert, in the presence of hydroxyl radicals, to oxidation products beyond t r a p p i n g by D M P O . W e therefore sought to assess whether o x i d a t i o n p r o d u c t s of hypotaurine might be detectable by direct c h r o m a t o graphy of the Fenton reaction mixtures where hydroxyl radical scavenging had previously been observed, but where, by EPR analysis, no t h i y I - D M P O a d d u c t s appeared to accumulate. Experiments were c o n d u c t e d by exposing stock 20 m M ~olutions of freshly prepared hypotaurine m a d e up in 41 m M p h o s p h a t e buffer (pH 7.8) also c o n t a i n i n g 83 m M E D T A to ferrous sulfate, hydrogen peroxide, and mixtures of ferrous sulfate and hydrogen peroxide. F o l l o w i n g a 15 rain i n c u b a t i o n at
lOG
C
lOG
Fig. 4. EPR spectralscansof DMPO products fcrmed by enzymatic peroaidation of cysteine, toluene sulfinate and hypotaurine. Thiyl substrate (final concentration 41 raM) in 41 mM phosphate buffer (pH 7.8), also supplemented with 0.83 mM EDTA and 71 mM DMPO was mixed with hydrogen peroxide (final concentration 3.9 raM) and horseraddish peroxidasc (final concentration 0.5 mg per ml). Scans were made approx. 8 rain after the addition of horseraddish peroxidase and hydrogen peroxide with EPR settings as in Fig. 2 except for the gain which was increased to I. 104. Spectral scans shown are with: a, cysteine; b, toluene sutfinate; c, hypotaurine; and d. control scan conducted in the absence of thiyl subs~rate.
95 room temperature, 10 to 20 #1 aliquots of the reaction mixtures were derivitized with o-phthaldialdehyde and immediately analyzed. By analysis of the relative differences in peak recoveries and retention times among the samples tested, two resolvable oxidation products of hypotaurine were detected. One of the reaction products was identified as taurine. The more polar product (suggested by its shorter retention time on the reverse-phase column) has not yet been identified. Chromatographic conditions were as follows for observation of these oxidation products: column starting buffer, 5% buffer B in buffer A held constant for the first 5 rain after injection of the sample at a flow rate of 0.9 ml per re!n:
I
lYr it
:~T
i "
'
I
.
!I
T,me (re,n)
o
T,me (m:n)
l i I, .T :i
I) iscu.~ion
I; '!
i
°
tI,.T !i
:i
i;
!
thereafter, the concentration of buffer B was increased by 1.4c~c per rain linearly to 100% buffer B; the column was re-equilibrated in starting mobile-phase buffer to 15 rain after holding at 100% B for 10 min. Fig. 5a shows the effect of incubating hypotaurine in phosphate-EDTA buffer free of ferrous sulfate and hydrogen peroxide compared to incubations conducted in the presence of hydrogen peroxide (Fig. 5b), ferrous sulfate (Fig. 5c), and a mixture of ferrous sulfate and hydrogen peroxide (Fig. 5d). liypotaurine was recovered in these HPLC separations at a retention time of approx. 40.3 rain. Taurine (present as a contaminant in the starting stock solution of hypotaurine at an identical concentration of al~prox. 0.9%) eluted with a retention time of 41.2 min. Upon exposing hypotaurine to ferrous sulfate the taurine level rose to 3.7%. lit the presence of hydrogen peroxide taurine rose to 2.2%. With hypotaurine exposed to ferrous sulfate and hydrogen peroxide, approx. 5.7% of the hypotaurine was converted into taurine, and an additional 7.5% was detected in the form of a polar compound eluting with a short retention time of approx. 6.4 rain (of. Fig. 5d).
li,
T~me[rain) Ttme (.T:inI Fig. 5. Detection of oxidauon products of hypotaurine following exposure to hydroxyl radicals. Hypotaurine (20 raM) was allowcd to react with hydrogen peroxide and ferrous sulfate for approx. 15 min as in Fig. 2, but wtth the omission of DMPO. Aliquots were then dehvitized with o-pthaldialdehyde as in Fig. I and subjected to HPLC separation as in Materials and Methods. The mobile phase was modified as follows: startin 8 buffer, 5% buffer B in buffer A held
constant for 5 win after injection; thereafter, increase buffer B by 1.4% per ntin at a fixed flow rate of 0.9 ml per rain to a final concentration of buffer B of 100% I = HPLC injection started; HT = hypotaurine; T = taurine; and U = unidentified oxidationproduct (a) Hypotaurine without exposure to hydrogen peroxide and ferrous sulfate. (b) Hypotaurine+ hydrogen peroxide alone. (c) Hypotaurine + ferrous sulfate alone. (d) Hypotaurine+ ferrous sulfate+ hydrogen peroxide (completereaction mixture).
The detection of hypotaurine in the neutrophil has not heretofore been reported. Neutrophils are known to elicit a respiratory burst response to extracellular stimuli culminating in the extracellular release of several oxidant species including superoxide, hydrogen peroxide, hypochlorous acid and, possibly, hydroxyl radicals ]16l~l. Wasil eta!. [!9] and Gfisham el al [7~]have presented data which indicates that taurine acts as ae antioxidant in quenching the oxidizing activity of hypochlorous acid. in the present study the concentration of intracellular hypotaurine was observed to decrease approx. 80% concomitant with conversion of resting neutrophils into actively respiring cells with opsonized zymosan. This suggests that the biological role of hypotaurine, like that of taurine, is linked to the release of oxidants accompanying activation of resting neutrophils. Taurine chloramine formation would be the expected endproduct of hypotautine oxidation [4]. Since little is understood regarding the mechanism of hydroxyl radical mediated oxidation of hypotaurine0 we chose to investigate this latter reaction by exposure of hypotaurine to hydroxyl radicals generated via Fenton chemistry to gain a clearer insight as to the fate of hydroxyl radical mediated oxidation of hypotaurine. As evident in the experiments summarized in Fig. 5, hypotaurine was converted to taurine under conditions in which hydroxyl radical generation would be expected to occur via a Fenton reaction. Hence. laurine formation was seen upon exposure of hypotaurine to either ferrous sulfate or a combination of ferrous sulfate and hydrogen peroxide. The formation of taurine by hydroxyl
96 radical mediated oxidation of hypotaurine most likely occurs by the formation of a thiyl radical intermediate tot) unstable to be trapped by DMPO. Since oxidation of the sulfur moeity by hydroxyl radict, l would be expected 1o lead to formation of thiyl radical, and because we have previously demonstrated by mass spectral analysis formation of disulfon_e intermediates of hypotaurine under these experimental conditions [6], we believe it is reasonable to conclude that the rate of thiyl radical dimerization exceeds that of thiyl-DMPO formation. This would explain why thiyl-DMPO adducts of hypotaurine were not seen when hypotaurine was oxidized by Fenton chemistry in the presence of DMPO (cf. Fig. 2). On theoretical grounds the disulfone intermediate would be expected to hydrolyze to hypotaurine and taurine. Thus hydroxyl radical mediated oxidation of hypotaurine leading to taurin*, formation likely progresses through the following series of reactions: hypotaurine + hydroxyl radical ~ sulfinyl radical
hydroxyl radicals, and since hypochlorous acid is a well-recognized product of respiring neutrophils, it is clear that measurements of hypotaurine turnover accompanying cell activation and expression of a respiratory burst response cannot serve as evidence that neutrophils produce hydroxyl radicals. However, it is possible that some oxidation products of hypotaurine, such as the disulfone intermediate, may be sufficiently specific products of hydroxyl radical mediated oxidation as to reflect its formation in neutrophils. In this respect, further work in charac*erizing specific h?droxyl radical mediated oxidation products of hypotaurine, and a search for their presence in respiring neutrophils. appears warranted. Studies along these lines should lead 1o a clearer understanding of the antioxidant role of hypotaurine and taurine in addition to the nature of the oxidants reaching the intracellular spaces of the neutrophil where these two unusual amino acids reside. Acknowledgements
2 sulfinyl radicals ---, disulfone disulfone + hydroxide ---, hypotaurine + taurine At the present time, insufficient product has been isolated from the HPLC column to ascertain whether or not the polar oxidation product shown in Fig. 5d is the disulfone intermediate. Hence, we do not know whether the polar peak is the a-disulfone intermediate, or a third, yet unidentified, oxidation product of hypotaurine. Further work will be required in determining its chemical structure before a search for hydroxyl radical mediated oxidation products of hypotaurine in respiring neutrophil suspensions can be conducted. Since neutrophils direct their oxidative activity extracellularly in killing bacteria, whereas hypotaurine and taurine clearly reside within the cytosol (cf. Table I), the oxidation of hypotaurine and taurine residing in the cytosolic compartment following cell stimulation may at first appear enigmatic. However, Albrich et al. [20] have shown that hypochlorous acid bleaches carotene embedded in bacterial cell walls. They interpreted these results as evidence that cell membranes pose no significant barrier to hypochlorous acid. Ohno et al. [21] have obtained cytochemical evidence that hydrogen peroxide formed by respiring neutrophils crosses the neutrophil membrane and enters the cytoplasmic space. The presence of hydrogen peroxide and transition metals in the cytoplasmic compartment may also allow for Fenton-type reactions to occur within the cytosol. Hence, on chemical grounds it appears that the neutrophil cytosol fraction is not free from oxidative assault. Since the chemistry of hypotaurine is such that it is readily oxidized by hypochlorous acid in addition to
This work was supported by the Medical Research Foundation of Oregon and the Veterans Administration. References
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