Free metal ion-independent oxidative damage of collagen. Protection by ascorbic acid.

In this paper we demonstrate that in the absence of free metal ions, active oxygen species, generated by activated macrophages or xanthine/xanthine oxidase (XOD), carry out oxidative degradation of collagen fibrils type I in conjunction with proteases. The collagen degradation is completely prevented by ascorbate (AH2) but not by catalase. The free metal ion-independent collagen degradation is a two-step process: (i) oxidation of collagen and (ii) subsequent proteolytic cleavage of the oxidatively modified collagen. AH2 completely prevents collagen oxidation and thereby protects the collagen from subsequent proteolytic degradation. This is in contrast to free metal ion-catalyzed spontaneous fragmentation of collagen, which is accelerated by AH2 and inhibited by catalase (Kato, Y., Uchida, K., and Kawakishi, S. (1992) J. Biol. Chem. 267, 23646-23651). Studies using xanthine/XOD and model polypeptides, namely, poly-L-Pro, poly-L-hydroxyproline, poly-L-Lys, and poly(Pro-Gly-Pro) indicate that although O2-. is needed along with XOD, oxidation of model polypeptides appears to be a direct function of XOD iron, which is also stimulated by cytochrome P450.

The abbreviations used are: AH,, ascorbic acid; X, xanthine; XOD, xanthine oxidase; PMA, phorbol myristate acetate; NBD-Cl, 12-(A" methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-y1)) chloride; Fmoc-C1, N49-fluoreny1)methoxycarbonyl chloride; HPLC, high performance liquid chromatography; PMSF, phenylmethylsulfonyl fluoride. oxidation system Cu(II)/H,O,, Kat0 et al. (11) observed that spontaneous oxidative fragmentation of collagen a n d model polypeptides including poly-L-Pro and (Pro-Pro-Gly),, occurred because of oxidation of proline residues and cleavage of the peptide bond Pro-Gly. However, the aforesaid in vitro studies with added iron or copper salts appear to have less relevance to the i n vivo situation where, in the normal physiological condition, most of the metals are not free and remain tightly bound with proteins. Preliminary studies indicate that active oxygen species, generated by xanthine/xanthine oxidase (XKOD) or stimulated macrophages, cause degradation of collagen (13)(14)(15). In this paper, we have used model in vitro systems containing stimulated macrophages as well as XKOD to investigate the mechanism of oxidative degradation of collagen i n t h e absence of added metal salts. It is known that stimulated macrophages undergo respiratory burst and release in the environment large quantities of 0, (16)(17)(18) and proteinases and collagenases. It is also known that XOD contains proteases (19). We present evidence to demonstrate that degradation of collagen involving 0; is a two-step process: (i) oxidative modification of collagen as evidenced by carbonyl formation and (ii) subsequent rapid degradation of the oxidatively modified collagen by proteinases and collagenases. We further demonstrate that in contrast to acceleration of free metal ion-catalyzed oxidative fragmentation of collagen by AH, , A H , completely prevents free metal ion-independent X/XOD or stimulated macrophagemediated collagen oxidation and thereby subsequent proteolytic degradation.
We have also used model polypeptides, namely, poly-L-proline, poly-L-hydroxyproline, poly(Pro-Gly-Pro), and poly-L-lysine, to elucidate the mechanism of XKODmediated oxidative damage.
lsolatzon of Macrophages-Male guinea pigs (350-400 g) were injected intraperitoneally with 10 ml of sterile mineral oil and exudates harvested 4 days later (22). The cell suspension was washed three times with Earle's balanced salt solution (22) by centrifugation at 700 x g for 10 min. Erythrocytes were eliminated by hypotonic lysis with 0.2% NaCl. The cells were suspended to a concentration of 1 x lo7 cells/ml in 5 mM glucose containing Krebs-Ringer phosphate buffer, pH 7.4, composed of 120 mM NaCI, 4.8 mM KCl, 0.5 mM CaCI,, 1.2 mM MgSO,, and 15.6 mM sodium phosphate solution.
Superoxide Generating Systems-In the macrophage system, 0; was generated by adding 1 pg of PMA to 1.5 ml of glucose/Krebs-Ringer phosphate buffer containing 1 x lo7 cells (preincubated at 37 "C for 90 s) and 10 p~ desferrioxamine. Desferrioxamine was added to chelate any contaminating free iron. The rate of generation of 0, was measured by determining the rate of superoxide dismutase-inhibitable ferricytochrome c reduction at 550 nm (23) in a double beam spectrophotometer (Hitachi model 200-20).
The XKOD system consisted of 0.1 M potassium phosphate buffer. 2H 7.4,5 x IO4 M xanthine, 10 PM desferrioxamine, 3 x ferricytochrome c, and 2 mM EDTAin a final volume of 1 ml. The reaction was started by the addition of requisite amounts of XOD, incubated at 37 "C. Superoxide production was measured by monitoring superoxide dismutaseinhibitable ferricytochrome c reduction (24).

Exposure of Collagen Fibrils to Activated Macrophages
and XiXOD-One mg of collagen fibrils (prewashed by centrifuging a t 12,000 x g for 15 min three times with buffer to remove any contaminating soluble peptides) was exposed to the macrophage (1 x lo' cells) system as stated above in a final volume of 1.5 ml except that ferricytochrome c was omitted. The mixture was preincubated at 37 "C for 90 s, and 1 pg of PMA was added to it to start generation of 0,. The incubation was continued at 37 "C.
In the XKOD system without ferricytochrome c, 1 mg of prewashed collagen fibrils was suspended in 0.1 M potassium phosphate buffer, pH 7.4, containing 5 x lo4 M xanthine, 10 PM desferrioxamine in a final volume of 1 ml. The reaction was started by the addition of requisite amounts of XOD, incubated a t 37 "C.
Measurement of Collagen Degradation-The degradation of collagen was measured by estimating both the release of hydroxyproline-containing soluble peptides and the liberation of fluorescamine-reactive materials (peptides with a new NH, terminus). After exposure of the collagen fibrils to the macrophage system or XKOD, as described ahove, the suspension was centrifuged a t 12,000 x g for 15 min, and the clear supernatant was analyzed for soluble hydroxyproline-containing peptides. Hydroxyproline was liberated from the peptides by hydrolysis with 6 N HCl under nitrogen for 16 h at 120 "C followed by evaporation aliquots of this solution were applied to a silica gel TLC plate, activated a t 65 "C for 10 min and developed using a solvent mixture containing methanol/toluene/acetone/triethylamine (15/40/40/5, v/v) as described elsewhere (25). A blank and a standard were run side by side. The fluorescence spots were eluted by a mixture of ethanoVwater (5060, v/v) and the fluorescence read setting excitation and emission wavelengths a t 340 and 525 nm, respectively, in a Hitachi fluorescence spectrophotometer model F-3010. NBD-hydroxyproline separated as a clear distinct spot without having any chance of contamination by NBD-Pro or other amino acids.
The hydroxyproline obtained by acid hydrolysis of soluble peptides after exposure of collagen fibrils to XKOD was also estimated by HPLC after derivatizing with Fmoc-C1 as described elsewhere (26). Twenty-pl aliquots of the Fmoc-C1 derivative of hydroxyproline were applied to a Shimadzu Shim-pack CLC-ODS(M) column connected to a Shimadzu UV detector and Shimadzu C-R6A Chromatopac. Elution was performed using a binary gradient of buffer A containing 100 mM sodium acetate trihydrate with 0.5% tetrahydrofuran and buffer B containing 80% acetonitrile and 20% 100 mM sodium acetate trihydrate, adjusted to pH 7.4. The absorbance was monitored a t 263 nm. Retention time of Fmoc-C1 derivative of hydroxyproline was approximately 14.5 min. The amount of hydroxyproline in the hydrolysates of soluble peptides was quantified from a standard curve.
Degradation of collagen fibrils after exposure to the X/XOD system was also measured by the production of fluorescamine-reactive material in the neutralized trichloroacetic acid-soluble supernatant (27). To 0.25 ml of neutralized supernatant was added 1.25 ml of 50 mM HEPES, pH 9.0, followed by dropwise addition of 0.5 ml of a 0.3 mg/ml fluorescarnine solution in acetone, while vortexing each time. The results were evaluated by fluorometry using a n excitation wavelength a t 390 nm and Effects of scavengers of reactive oxygen species on the oxidative degradation of collagen after exposure to activated macrophages and XIXOD In the PMA-activated macrophage system, the incubation medium contained 1 mg of calf skin collagen fibrils (-800 nmol of hydroxyproline), 10 PM desferrioxamine in a final volume of 1.5 ml of Krebs-Ringer phosphate buffer, pH 7.4, containing 5 mM glucose; incubated at 37 "C for 1 h. The amount of 0; produced during the initial 5-8 min was 100 rophages, 1 x lo7 cells; PMA, 1 pg; ascorbic acid, 20 p; superoxide f 10 nmol. Additions were made at the following concentrations: macdismutase, 100 units; catalase, 40 pg; mannitol, 20 mM; thiourea, 10 mM; histidine, 10 mM. Hydroxyproline was estimated by TLC-fluorometry as described under "Experimental Procedures." In the XKOD system, the incubation medium contained 1 mg of calf skin collagen fibrils, 0.9 ml of 0.1 M potassium phosphate buffer, pH 7.4, 10 p~ desferrioxamine, 5 x M xanthine, and 100 milliunits of XOD in a final volume of 1 ml. Other additions were same as in the macrophage system stated above. The amount of 0, produced during the initial 5-8 min was 100 * Assay of Carbonyl Content-The carbonyl content of collagen produced by oxidative modification was estimated by the method of Levine et al. (28). One mg of collagen fibrils was exposed to the XKOD system in the presence of 100 pg of PMSF for 15 min. PMSF was added to prevent proteolytic degradation of oxidized collagen by proteases present in the sample of XOD used (19). After incubation, the suspension was centrifuged at 12,000 x g for 15 min. The supernatant was discarded, and the sedimented collagen fibrils were washed twice with phosphate buffer. The carbonyl content of the oxidatively modified collagen was estimated by reaction with 2,4-dinitrophenylhydrazine (28).

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis f'SDS-PAGE)-PAGE
(12% gel) in the presence of 0.1% SDS was performed according to the method of Laemmli (29). The gels were silver stained (30).
Estimation of Iron-Contaminating free iron contents of XOD, reagents, and buffer were estimated by the desferrioxamine binding assay (31).
Oxidative Modification of Model Polypeptides-One mg each of the model polypeptides, namely, poly-L-Pro, poly-L-hydroxyproline, poly-^-Lys, and poly(Pro-Gly-Pro), was added to 0.1 M potassium phosphate buffer, pH 7.4, containing 5 x lo-, M xanthine, 10 PM desferrioxamine in a final volume of 500 pl. The reaction was started by the addition of requisite amounts of XOD and incubated at 37 "C for 15 min. Fluorescamine-reactive material was analyzed in the trichloroacetic acidsoluble fraction of the incubated mixture (27). To estimate the carbonyl content of the oxidized polypeptides, 50 pg of PMSF was added to the incubation mixture to prevent proteolytic degradation of the oxidized polypeptides. The carbonyl content was assayed in the trichloroacetic acid-precipitated fraction of the polypeptides (28).

RESULTS
Collagen Degradation by Activated Macrophages-When guinea pig intraperitoneal macrophages (1 x lo7 cells) were activated with 1 pg of PMA, about 100 nmol of 0; was released in the medium. Exposure of collagen fibrils (1 mg) to the PMAactivated macrophages resulted in rapid degradation of collagen, as evidenced by the release of hydroxyproline-containing soluble peptides in the medium (Table I) Table I, collagen degradation was completely inhibited by 100 units of superoxide dismutase or 20 p~ AH, . Catalase, mannitol, thiourea, and histidine were ineffective, indicating that 0, but not H,O,, 'OH, or lo, was involved in collagen degradation. Macrophages without activation by PMA, sonicated macrophages + PMA, or cell-free extract of PMA-activated macrophages failed to degrade collagen fibrils. Degradation of Collagen afrer Exposure to XIXOD System-Exposure of collagen fibrils to the XKOD system also resulted in rapid degradation of collagen, as evidenced by the liberation of soluble hydroxyproline-containing peptides (Table I). In this case, hydroxyproline was estimated both by TLC-fluorometry and HPLC. The values were similar. As observed in the case of PMA-activated macrophages, collagen degradation was completely prevented by 100 units of superoxide dismutase or 20 PM AH, . Higher concentrations of AH, (up to 100 p d produced similar inhibition. Only collagen + XOD or collagen + xanthine did not cause degradation. It is known that the XKOD system generates not only 0, but also H,O,. However, degradation of collagen exposed to XKOD was not inhibited by catalase. Mannitol, thiourea, and histidine were also ineffective. These results again indicate that 0, and not H,O,, 'OH, or '0, was involved in oxidative degradation of collagen in the XKOD system. Role of Iron in Collagen Degradation-Superoxide has been considered to be chemically not that reactive (81, and direct involvement of 0, in protein oxidation has not been substantiated (7). We observed that omission of desferrioxamine from the XKOD system resulted in a 47% increased degradation of collagen over that obtained in the presence of desferrioxamine. The desferrioxamine binding assay (31) indicated the presence of about 1 VM adventitious iron in the reagents and buffers of the incubation mixture and 3.1 p~ free contaminating iron in the sample of XOD used. Apparently, this contaminating iron probably caused the increased collagen breakdown in the absence of desferrioxamine. The increase in collagen breakdown in the absence of desferrioxamine was inhibited by catalase but not by AH, . Also, the addition of ADP-Fe3+ to the incubation medium (in the absence of desferrioxamine) resulted in a further increase (90%) in collagen fragmentation. Again, catalase inhibited this increased degradation, but A H , failed. These results indicate that in the presence of contaminating free iron or added ADP-Fe3+, a fraction of the collagen degradation is mediated by H,O,, as visualized by Stadtman and Oliver (1) in the metal-catalyzed protein oxidation. We consider that oxidative degradation of collagen involving 0; is probably mediated by protein-bound redox iron present in XOD (32) or that secreted by activated phagocytes (33). It is known that the XOD molecule contains four iron centers having redox property (32) and that activated phagocytes secrete cytochrome b,,, (33). It is also known that desferrioxamine is incapable of chelating large polypeptide-bound iron (34).
In addition to assessing collagen degradation by measuring the generation of hydroxyproline-containing soluble peptides, degradation of collagen was also evidenced by the production of trichloroacetic acid-soluble fluorescamine-reactive peptides. Fluorescamine reacts with NH, groups. Production of fluorescamine-reactive materials indicates the production of new NH,-terminal peptides. After exposure of collagen fibrils to the XKOD system, the production of fluorescamine-reactive material continued for 60 min (Fig. 1). In the presence of desferrioxamine, A H , completely prevented the production of fluorescamine-reactive materials. Catalase, mannitol, thiourea, and histidine were ineffective (data not shown). On the other hand,  Tables I and 11. After incubation, the mixture was deproteinized with 10% trichloroacetic acid. As mentioned under "Experimental Procedures," 0.25-ml aliquots of neutralized trichloroacetic acid-soluble supernatants were used for the fluorescamine test. The fluorescamine unit (154) obtained after incubation of collagen fibrils in the absence of xanthine but in the presence of XOD was deducted from all experimen-S.D. < 10%. 0, ADP-Fe3+ plus AH,; 0 , ADP-Fe3+; A, in the absence of tal data. Values are the means of three independent determinations; ADP-Fe3+; x, in the absence of ADP-Fe3+ plus AH,.
when ADP-Fe3+ was added to the incubation medium, the production of fluorescamine-reactive material increased about double, and A H , failed to prevent ADP-Fe3+-mediated collagen breakdown (Fig. 1). These results confirm that there are two distinct mechanisms of oxidative degradation of collagen: (i) mediation by protein-bound redox iron and completely inhibitable by A H , and (ii) catalysis by free iron ions and stimulated Involvement of both 0, and Proteinases in Collagen Degradation-After the addition of PMA to the macrophage system, 0, production continued only for the initial 5-8 min after which no further 0, was produced. Superoxide production was monitored by the reduction of ferricytochrome c added initially to a separate but identical incubation medium. When ferricytochrome c was added 10 min after the addition of PMA, no reduction of ferricytochrome c was noticed, indicating that 0, production virtually stopped. Although 0, production stopped, degradation of collagen continued almost linearly for at least 60 min. This indicates that 0, is probably needed initially for oxidative modification of collagen and that the modified collagen is then probably hydrolyzed by proteinases and collagenases released from PMA-activated macrophages (18). This was substantiated by the fact that the addition of inhibitors of proteinases and collagenases, namely, PMSF (100 pg) and EDTA (20 mM), 15 min after the addition of PMA prevented further release of hydroxyproline-containing soluble peptides. Similar observation was made in the =OD system where PMSF (100 pg) added initially to the incubation mixture almost completely inhibited collagen degradation. PMSF did not inhibit 0, production by XKOD. The proteolytic cleavage in the =OD system was apparently carried out by contaminating proteases present in XOD (19). No hydroxyproline was released within the short span of the 1-h incubation period when untreated collagen was incubated with proteinases and collagenases. Once the collagen was oxidatively modified, A H , or by AH,.

T,wI.F: I1
Effect of AH, on the oxidation of collagen h.y X I X O D One mg of collagen was exposed to about 100 nmol of 0; in X/XOD system in the presence of 10 p~ desferrioxamine and PMSF (100 pg) in a 1-ml system as described in Table I except that the concentration

Oxidative Modification of Collagen as Evidenced by the Introduction. of Carbonyl Groups-Measurement
of carbonyl groups is a sensitive assay for assessing oxidative modification of proteins (28). We have estimated carbonyl groups by reaction with 2,4-dinitrophenylhydrazine (28). Table I1 shows that treatment of collagen fibrils with XKOD results in a large increase of hydrazone formation. Since XOD contains contaminating proteinases, we have used PMSF to prevent proteolytic degradation of oxidatively modified protein and thereby loss of carbonyl in the soluble peptides. Hydrazone was not produced when collagen was treated with either xanthine or XOD. Table  I1 further shows that hydrazone formation was completely inhibited by AH, or superoxide dismutase but not by catalase.
That A H , inhibits XKOD-mediated oxidative modification of protein has also been reported by others (35). However, when ADP-Fe3+ was added to the incubation medium in the absence of desferrioxamine, collagen oxidation was 74% stimulated by AH,, indicating that in the presence of ADP-Fe"' A H , acted as a prooxidant. Fig. 2 shows that carbonyl formation in collagen is a function of XOD concentration. The amount of carbonyl increases linearly with increased concentration ofXOD from 0.1 to 0.5 nmol studied. However, as shown in Table 11, XOD alone cannot oxidize collagen. Superoxide is needed along with XOD because XKOD-initiated collagen oxidation is completely prevented by superoxide dismutase. Moreover, carbonyl formation virtually stops after 10 min of incubation, when 0; generation also ceases. Although collagen oxidation is dependent on 0, formation, the amount of carbonyl formation does not appear to be stoichiometrically related to 0; concentration, because even with 0.1 nmol of XOD, 20 nmol of 0, is generated, which is theoretically more than sufficient to produce the maximum amount of carbonyl (2 nmol) formed (Fig. 2).
Oxidatively Modified Collagen Is Readily Hydrolyzed hy Proteinases and Collagenases-It is known that oxidized proteins are highly susceptible to proteolytic degradation (1)(2)(3)(4)(5)(6)(7)(8)(9). This has also been confirmed for collagen by SDS-PAGE of native and oxidatively modified collagen fibrils (Fig. 3). Fig. 3 shows that oxidized collagen isolated after pretreatment with XKOD undergoes random hydrolysis by trypsin + chymotrypsin, produc-  Table 11. A basal value of 0.68 0.09 obtained with native collagen was deducted from the experimental data. Carbonyl groups were estimated after 10 min of incubation. Prolonged incubation did not produced further hydrazone formation. Results are the means of four independent determinations; S.D. < 10%.  Table 11) were incubated with trypsin (50 ug) plus chymotrypsin (25 pg) in 1 ml of 0.1 11 potassium phosphate buffer, pH 5.4, for 1 h at 37 "C. After incubation, the mixture was centrifuged, sedimented collagen discarded, and the supernatant freeze-dried and dissolved in 50 pl of water. Five p1 of this solution was subjected to 12% SDS-PAGE. ing a number of low molecular weight peptides ranging from 14,000 to 90,000. Native collagen is not hydrolyzed. Also, no hydrolysis is obtained when XKOD treatment of collagen fibrils is done in the presence of AH, (50 p r ) , which further confirms that A H , prevents oxidative damage of collagen. One mg of each polypeptide was exposed to about 100 nmol of 0, in the X/XOD system. Other conditions are same as in Fig. 2 The collagen helix contains the characteristic repeating sequence of Gly-X-Y, where X and Y are often Pro and hydroxyproline, respectively. We have studied oxidation of poly-t-Pro, poly-L-hydroxyproline, and the collagen-like sequence, namely, poly(Pro-Gly-Pro). Type I collagen contains about 4% Lys (36), so we have also studied oxidation of PO~Y-L-LYS. We have observed that oxidation of poly-L-Pro/poly-L-Lys/poly(Pro-Gly-Pro) by =OD in the presence of desferrioxamine is completely inhibited by A H , (Table 111). The oxidation was assayed by carbonyl formation.
In contrast to this, when ADP-Fe3+ was added along with XKOD in the absence of desferrioxamine, the oxidation of poly-L-Pro was 100% enhanced, and it was further stimulated (128%) by AH, . The results again confirm that in the absence of metal ions A H , acts as an antioxidant, but in the presence of ADP-Fe3+ it acts as a prooxidant. We have further observed that in contrast to poly-L-Pro, poly-L-hydroxyproline is not oxidized by =OD.
Oxidation of Model Polypeptides as a Function of the Concentration of Protein-bound Redox Iron-It has been indicated that although 0, is needed along with XOD for collagen oxidation, carbonyl formation in collagen is a function of XOD concentration (Fig. 2). This has been substantiated further by our work with poly-L-Pro and poly(Pro-Gly-Pro). Using =OD as a source of 0, and keeping the amount of 0, production limited (5)(6)(7)(8)(9) nmol) with a limiting concentration of xanthine (25 pM), the formation of carbonyl in poly-L-Pro appears to be a direct function of the concentration of XOD iron. Fig. 4 shows that carbonyl formation in poly-L-Pro increases linearly with increased concentration of XOD iron. Cytochrome P450 is another enzyme that is known to contain protein-bound iron having redox property (37). Fig. 4 further shows that keeping the amounts of XOD (0.4 nanoatom of iron) and 0; (5 nmol) constant and adding increased amounts of cytochrome P450 in the medium result in a linear increase of carbonyl formation. A similar observation has been made using poly(Pro-Gly-Pro) in the place of poly-L-Pro (Fig. 5). However, ferritin (20 PM) or hemoglobin (2 p~) could not replace cytochrome P450.

DISCUSSION
In the present investigation, we have demonstrated that in the absence of free metal ions active oxygen species, generated by activated macrophages or XKOD, carry out oxidative degradation of calf skin collagen fibrils in conjunction with proteases. The triple helical structure of collagen, particularly type I, is normally resistant to the action of most proteases. However, once the collagen is oxidatively modified, the fibrils become highly susceptible to proteolytic degradation. This is consistent with the view that oxidatively modified proteins are highly sensitive to proteolytic breakdown (1)(2)(3)(4)(5)(6)(7)(8)(9). Although free metal ion-catalyzed oxidation of collagen has provided valuable information about the mechanism of oxidative fragmentation of collagen (ll), its relevance to the in vivo situation is questionable, apparently because in the normal physiological condition most of the metal ions are not free and remain tightly bound with proteins. The mechanism of free metal ion-independent collagen oxidation is distinctly different from that catalyzed by CutII)/H,O,. The free metal ion-independent collagen degradation is a two-step process: (i) oxidation of collagen and (ii) subsequent proteolytic cleavage of the oxidatively modified collagen. In the absence of free metal ions, AH, completely prevents oxidation of collagen and thereby protects the collagen from subsequent proteolytic degradation. Catalase is ineffective. This is in contrast to Cu(II)/H,O,-catalyzed spontaneous oxidative fragmentation of collagen which is accelerated by A H , but inhibited by catalase (11).
It has been shown by Davies and co-workers (7, 9) that 0, alone, generated by 6oCo radiation, does not significantly damage proteins. We have observed that collagen oxidation by XKOD is rather a function of protein-bound redox iron of XOD. XOD (EC 1.1.3.22) is a homodimer with a molecular weight of 300,000 having one molybdenum center, two iron-sulfur centers, and one molecule of FAD per subunit (38). It has been reported (38) that during 0, production (0, reduction) the ironsulfur centers of XOD are transiently reduced in the course of electron transfer from the molybdenum center to the flavin. Redox iron is also probably involved in collagen oxidation by activated macrophages, which are known to secrete in the medium cytochrome b558 (33). For collagen oxidation, only XOD is ineffective. XKOD-mediated oxidative modification of collagen is completely inhibited by superoxide dismutase. This indicates that both protein-bound redox iron and 0: are needed to prod- uce an active oxygen species for collagen oxidation. The involvement of redox iron has been substantiated by our work with model polypeptides. Oxidations of poly-L-Pro and poly(Pr0-Gly-Pro) have been found to be a direct function of the concentration of XOD iron. Cytochrome P450 also stimulates this oxidation.
If the results obtained in vitro with activated macrophages are applicable to the in uivo situation, then our results may throw some light on the poorly understood mechanism of collagen degradation in scurvy and protection by AH,. The extracellular matrix of mammals contains numerous macrophages that undergo oxidative burst during phagocytosis and release in the environment large amounts of 0, (161, redox protein like cytochrome b558 (331, as well as metalloproteinases and collagenases (18). We have presented evidence that activated macrophages carry out oxidative degradation of collagen and that A H , completely prevents this degradation. Superoxide dismutase also prevents it, but in contrast to A H , which is ubiquitous in uiuo, the content of superoxide dismutase in the extracellular fluid is negligible (39). Only a small amount of ttive Damage of Collagen 30205 glycosylated tetrameric superoxide dismutase is present in the extracellular fluid (40). This imparts a specific important role of A H , for the protection of collagen in the extracellular matrix of mammalian tissues.