BACKGROUND
members of the genus prevotella are associated with periodontal diseases in humans and other animals  <cit> .
they have a growth requirement for iron protoporphyrin ix, and display a characteristic black-pigmenting phenotype due to cell-surface accumulation of haem  protoporphyrin ix) in the monomeric form ppix.oh, haematin), derived via the breakdown of haemoglobin  <cit> .
the haem-containing pigments of both prevotella and porphyromonas species are important defensively, since ferrihaems in both the monomeric and dimeric ppix]2o) forms possess inherent catalase activity and thus can protect against hydrogen peroxide  <cit>  which is produced by activated neutrophils.
in the primary habitats of the black-pigmenting anaerobes , the most abundant haem source is haemoglobin.
however, in healthy or minimally inflamed gingival crevices, in the absence of overt bleeding , their survival and growth may depend on haemalbumin and haem-haemopexin as primary haem sources.
these proteins normally sequester any circulating free haem and transport it to the liver  <cit> , thus limiting its bioavailability to invading micro-organisms.
albumin is the most predominant protein from both healthy and inflamed periodontal disease sites .
although albumin has a 75-fold lower affinity for haem compared to haemopexin  <cit> , its haem sequestering capability is compensated by its 40-80-fold greater concentration in serum  <cit> , and therefore it binds the majority of any haem liberated into the circulation.
in addition, in normal serum, approximately  <dig> in  <dig> albumin molecules is haem liganded  <cit> .
importantly, these haemalbumin molecules may serve as a non-haemoglobin source of iron protoporphyrin ix under conditions of haem limitation for p. intermedia and other sub- and supra-gingival plaque bacteria.
we have investigated the mechanism of protease-mediated haem release from haemoglobin by p. intermedia  <cit> .
this organism expresses a 90-kda cysteine protease, termed interpain a   <cit> , an orthologue of periodontain  of p. gingivalis and streptopain  of streptococcus pyogenes  <cit> .
inpa is likely to serve a major role in pigment production through oxidising oxyhaemoglobin and then fully degrading the methaemoglobin product to release haem  <cit> .
in addition, it has been recently shown that inpa may serve to aid p. gingivalis haem acquisition by proteolytically mediating formation of methaemoglobin from which the p. gingivalis hmuy haemophore can extract haem  <cit> .
cell associated protease activity of p. intermedia can degrade albumin and haemopexin  <cit> , although the proteases mediating this have not been characterised.
incubation of the bacterium with albumin intensifies its proteolytic activity and growth  <cit> .
given that albumin represents an important source of haem under conditions of haem scarcity, we examined the ability of inpa to degrade albumin in both its apo- and haem-liganded forms under conditions which may prevail in the primary habitats of this bacterium, the inflamed gingival sulcus and diseased periodontal pocket.
RESULTS
since the redox potential of diseased periodontal pockets can be as low as − <dig> mv  <cit>  and that p. intermedia may experience wide fluctuations of ph within dental plaque, it was considered appropriate to examine the effect of these two variables on the activity of inpa.
our previous study  <cit>  showed that inpa activity was stimulated by dithiothreitol  and so we examined the effect of various concentrations of dtt on inpa activity versus the fluorescent synthetic substrate z-arg-arg-amc .
this revealed that enzyme activity began to plateau at  <dig> mm .
we then determined the influence of the thiol reducing agents cysteine, reduced glutathione and β-mercaptoethanol  on inpa activity using the above substrate, and this showed that dtt gave the greatest stimulatory effect .
however, in subsequent experiments to examine the influence of reducing conditions on albumin and haemalbumin degradation, we employed  <dig> mm dtt to ensure full enzyme activation.fig.
<dig> effect of dithiothreitol  and other thiol reducing agents  on the enzyme activity of inpa.
the enzyme activity was assayed in triplicate using z-arg-arg-amc at  <dig> °c in  <dig> m tris–hcl, ph  <dig>  and the concentrations of the reducing agents in fig.
1b were  <dig> mm.
due to the high reproducibility of the replicates, no sd is apparent.
control, activity in the absence of reducing agent.
inpa concentrations in  and  were  <dig>  μg ml− <dig>  see text for details
inpa displayed a broad activity against z-arg-arg-amc over the ph range from  <dig> to  <dig> .
however, for azo-albumin, the maximal activity was centred at ph  <dig> , with little or no activity beyond ph  <dig> or above  <dig> .
this may be important in terms of protein breakdown since p. intermedia can generate acid during growth in both liquid and solid media  <cit> , and may thus depress the ph locally in vivo.
we also investigated the temperature activity profile for inpa and thermal stability of the enzyme using azocoll and the synthetic substrate z-arg-arg-amc, respectively.
the enzyme showed maximal activity in the temperature range from  <dig> to  <dig> °c versus azocoll .
however, the enzymatic activity was found to be drastically decreased after the protein was pre-incubated for  <dig> min at temperatures above  <dig> °c, followed by assay with z-arg-arg-amc .
this indicates that inpa should be highly active at elevated temperatures which may be encountered in vivo in the inflamed gingival sulcus or periodontal pocket during periods of inflammation.
.fig.
<dig> effect of ph on inpa activity towards z-arg-arg-amc  and azoalbumin .
protease activity was assayed at  <dig> °c in pbs, ph  <dig> , containing  <dig> mm dtt.
inpa concentration was  <dig> μg ml− <dig>  azoalbumin concentration was  <dig> % .
enzyme activity is depicted as absorbance of azo dye  released from the protein.
see text for details
in the absence of dtt, there was little or no degradation of albumin , and densitometry showed that total apoalbumin degradation over  <dig> h was less than  <dig> % at all three phs studied.
in contrast however, for haemalbumin, three lower molecular weight peptide fragments of rf  <dig> ,  <dig>  and  <dig> , were generated at ph  <dig>  and  <dig>  .
the two fastest migrating of these bands, which stained with tmb-h2o <dig> and hence retained bound haem, were further degraded with time and also showed progressive loss of haem staining, as did the main 66-kda haemalbumin band, especially at ph  <dig>  after  <dig> h. little protein degradation was observed at ph  <dig> .
however, after  <dig> h, tmb-h2o <dig> staining of haemalbumin had decreased by circa  <dig> % at all phs examined, showing that extensive proteolysis was not necessary to effect haem release from the molecule.
in contrast however, the percentage of haemalbumin protein degraded after  <dig> h was greater at ph  <dig>  , compared to either ph  <dig>   or ph  <dig>   .
it should be noted that both apo- and haemalbumin were stable over  <dig> h at all ph conditions tested in the absence of inpa .fig.
<dig> albumin and haemalbumin breakdown by inpa at different ph under non-reducing conditions.
albumin and haemalbumin   were incubated with inpa  at  <dig> °c and aliquots of the incubations were sampled periodically at  <dig>   <dig>   <dig>   <dig> and  <dig> h and inpa activity inhibited by e- <dig>  and subjected to sds-page on  <dig> % non-reducing gels.
samples were solubilised at  <dig> °c for  <dig> h in non-reducing application buffer.
the haemalbumin gels were stained with tmb to reveal haem-associated peroxidise activity and counterstained with cbb for protein, whilst albumin gels were stained only with cbb.
protein loading was ≈  <dig> μg per lane.
arrows denote digestion fragments carrying bound haem
despite inpa displaying optimum activity towards azo-albumin at ph  <dig>   and being more active against aquomethaemoglobin at this ph  <cit> , it is interesting that haemalbumin degradation by inpa was negligible at ph  <dig>  but greatest at the slightly alkaline ph of  <dig> .
this apparent increase may not reside in inpa activity per se, but may result from structural changes in the albumin molecule occurring in solution at high ph  <cit> .
albumin displays a greater thermal stability at ph  <dig>  than at ph  <dig>   <cit> , undergoing a conformational change around ph  <dig>  referred to as the n → b transition  <cit> , pointing to the b form being more susceptible to inpa, as has been demonstrated for trypsin  <cit> .
this may be physiologically relevant as diseased periodontal pockets and the inflamed gingival sulcus are slightly alkaline  <cit> .
however, sds-page revealed  that haem loss also occurred at acid ph despite the low level of proteolysis, suggesting that only limited proteolysis by inpa is required to loosen the haem-albumin association.
compared to apoalbumin, haemalbumin is more susceptible to inpa.
haem complexing to bovine serum albumin also leads to greater proteolysis by trypsin and chymotrypsin  <cit>  although proteolysis of haemalbumin by whole cells of p. gingivalis is decreased, particularly as the ratio of bound haem to protein increases  <cit> .
nevertheless, conformational changes occurring upon haem ligation  <cit>  may account for inpa attack, enabling inpa to differentiate between apoalbumin and the scarcer haem-liganded form, which represents approximately 1/ <dig> of the total albumin molecules in serum under normal physiological conditions  <cit> .
in stark contrast to non-reducing conditions, albumin degradation was greatly increased under reducing conditions  at all phs examined, but especially at ph  <dig> , where complete breakdown was observed after  <dig> h .
the presence of dtt also increased albumin susceptibility at ph  <dig>  .
however, at ph  <dig>  less than  <dig> % was broken down even when dtt was present.
albumin was found to be stable under reducing conditions in the absence of inpa as judged by minimal depreciation in cbb staining observed only after  <dig> h incubation at ph  <dig>  .
in contrast to reducing conditions, apoalbumin was less susceptible to inpa , and densitometric scans  indicated that approximately  <dig> % had been degraded after  <dig> h at each ph.fig.
<dig> chemical reduction increases the susceptibility of albumin to inpa.
albumin  was incubated with inpa  at different ph at  <dig> °c under non-reducing  and reducing conditions  .
gel lanes had a nominal loading of  <dig> μg albumin, and are stained with cbb.
samples were prepared and gels run as described for fig.
3
fig.
<dig> sds-page showing the stability of albumin and haemalbumin under reducing conditions at different ph.
albumin and haemalbumin  were reduced with  <dig> mm dtt and incubated at  <dig> °c.
sds-page conditions were the same as those in fig.
<dig>  the gels were stained with cbb
in view the above, and the compositional similarities between gingival crevicular fluid and plasma  <cit> , it was appropriate to examine the effect of inpa on whole blood plasma, under conditions commonly reflecting those in the inflamed sulcus and periodontal pocket i.e., alkaline ph and low redox potential.
sds-page revealed that overnight incubation of whole human plasma with inpa at ph  <dig>  and in the presence of  <dig> mm dtt, resulted in almost complete breakdown of the albumin band , whilst leaving many other plasma proteins relatively undigested.
in comparison, proteinase k, a broad spectrum serine endopeptidase without any peptide bond preference, degraded a large number of the proteins in whole plasma but was less efficient at degrading the albumin component when compared to inpa at the same enzyme concentration .
these data show that inpa is able to target albumin present in plasma.fig.
<dig> sds-page analysis of the breakdown of whole human plasma by inpa.
plasma , was incubated overnight at  <dig> °c in pbs, ph  <dig> , containing  <dig> mm dtt in absence  or presence  of active inpa  in a final volume of  <dig> μl.
~  <dig> μg total protein was loaded per lane.
arrow denotes albumin band.
samples were solubilised for  <dig> min at  <dig> °c in reducing application buffer, and gels were stained with cbb
fig.
<dig> comparative sds-page analysis of the digestion of whole human plasma by inpa and proteinase k. whole human plasma  was incubated overnight with inpa and proteinase k  at the indicated concentrations  at  <dig> °c in pbs, ph  <dig>  containing  <dig> mm dtt.
last lane of each gel was loaded with  <dig> μg of either inpa or proteinase k only.
samples were prepared and gels stained as in fig.
6
the increased susceptibility of albumin to inpa in the presence of dtt may arise from structural changes brought about by chemical reduction of the disulphide bridges.
conformational changes of the local tryptophan environment in albumin are reflected in fluctuations of intrinsic fluorescence  <cit> .
when apoalbumin was treated with dtt there was a reduction of fluorescence intensity and moderate shift of λmax, indicating a change in the protein structure .
fluorescent emission was not detected above  <dig> μm dtt, indicating that the concentrations used in the digestions  were sufficient to induce structural alterations in albumin.
since inpa was pre-activated with an excess of dtt, it is likely that this increased susceptibility of albumin to inpa under reducing conditions was due to structural changes in the substrate rather than to any over-stimulatory effect of dtt on protease activity per se.fig.
<dig> intrinsic fluorescence spectra of bovine albumin in the presence of dtt.
albumin  was treated with increasing concentrations of dtt in  <dig>  m nacl,  <dig>  m tris–hcl buffer, ph  <dig> , at  <dig> °c, and the fluorescence emission monitored after excitation at  <dig> nm
the low redox potential of diseased periodontal pockets is attributable to the presence of volatile sulphur compounds  including h2s and methyl mercaptan, generated by organisms including p. intermedia, p. gingivalis and fusobacterium nucleatum  <cit> .
p. intermedia is also recovered from periodontal disease sites of patients suffering from oral malodour linked to vsc production  <cit> .
the  <dig> disulphide bridges of albumin are sensitive to reduction, which stimulates tertiary structural changes  <cit> , leading to increased molecular flexibility and enhanced susceptibility to pepsin  <cit> .
chemical reduction resulting from bacterial metabolism occurs during batch-wise enrichment culture of sub-gingival plaque in serum, where redox potentials of - <dig> to − <dig> mv have been recorded, and correlate with both h2s production and enrichment in numbers of p. intermedia  <cit> .
since  <dig> mm dtt was sufficient for optimal inpa activity, and is a concentration which can depress the redox potential to between − <dig> and − <dig> mv in e. coli growth medium  <cit> , it is likely that the reducing environment of deep periodontal pockets also results in structural changes in albumin because of disulphide bond disruption  <cit> , rendering albumin highly susceptible to inpa.
predictably therefore, treatment of albumin with  <dig> mm dtt greatly increased degradation by inpa, especially at ph  <dig> .
the minimal breakdown at acid ph with dtt present, where inpa activity towards azo-albumin and aquomethaemoglobin is highest  <cit> , suggests that ph-dependent conformational changes  <cit>  may dictate the effectiveness of proteolysis by inpa.
it is noteworthy also that accessibility of reducing agents to disulfide bonds in albumin  increases above ph  <dig>   <cit> .
in the presence of  <dig> mm dtt the susceptibility of haemalbumin  to inpa was also dramatically increased .
this was especially noticeable at ph  <dig> ; an observation explained by the fact that haemin binding to albumin expands sub-domain ib  which may render other regions of the peptide chain available for attack.
to further assess the ability of inpa to bring about proteolytic haem release from albumin at ph  <dig>  haemalbumin conjugated agarose was used as a substrate.
after  <dig> h in the presence of dtt, inpa liberated approximately 3-fold more haem than under non-reducing conditions .
in this context it is noteworthy that the albumin-haem affinity is lowered under reducing conditions, which in vivo favours haem transfer to haemopexin  <cit> .
furthermore, we observed that under reducing conditions haemalbumin was intrinsically more unstable than its non-liganded counterpart, particularly at alkaline ph , which may explain its heightened susceptibility to enzymic digestion.
thus, should the local plaque or periodontal pocket redox potential decline, the additional effect of reducing agents in facilitating haem liberation may supplement inpa-mediated haem release.fig.
<dig> effect of reducing conditions on breakdown of haemalbumin by inpa at different ph.
haemalbumin  with a 1: <dig> haem to protein ratio was incubated at  <dig> °c with inpa  in the absence  and presence of  <dig> mm dtt .
samples and gels were run under non-reducing conditions on  <dig> % gels and stained for protein with cbb.
each gel lane had a nominal loading of  <dig> μg albumin
it is noteworthy that grenier et al.
<cit>  demonstrated albumin breakdown by p. gingivalis r- and k-gingipains in the presence of  <dig> mm dtt.
we have observed that albumin and haemalbumin are resistant to active forms of both hrgpa and kgp under non-reducing conditions .
these and the above findings highlight the importance of reducing conditions which prevail in the diseased periodontal pocket and which are essential for disrupting both the haem liganded and apoalbumin structures.
inpa has specificity for k, r, a and f residues at the p <dig> position of hydrolysed peptide bonds  and this was seen in the large number of peptide fragments generated under both reducing and non-reducing conditions .
in all,  <dig> inpa scission sites were exclusive to reducing conditions and  <dig> to non-reducing conditions, with  <dig> sites common to digestions under both incubation conditions.
the formation of more fragments in the presence of dtt is not surprising, as it may reflect the exposure of more susceptible cleavage sites at previously inaccessible residues resulting from disulphide bond disruption.fig.
<dig> primary sequence showing all the cleavage sites inflicted during incubation of bovine serum albumin with inpa.
albumin  was incubated at  <dig> °c with inpa  in  <dig> mm tris buffer, ph  <dig> , in the presence or absence of 2mm dtt, and the incubation mixture sampled over time subjected to maldi-tof mass spectrometry.
inpa scission sites exclusive to reducing () , non-reducing (), and to both incubation conditions () are indicated.
see text for details
bleeding, and consequently increased levels of haemoglobin and hence haem, is associated clinically with the inflamed gingival crevice and diseased periodontal pocket, where the host responds to bacterial presence by increasing levels of plasma stress proteins including haemopexin and albumin  <cit> .
in addition, the combined haemolytic and proteolytic activities of other sub-gingival micro-organisms may also transiently elevate local haem levels resulting in greater haem-albumin concentrations.
however, we show that inpa not only degrades apoalbumin which normally reduces the potential for haem-induced inflammatory events  <cit>  and limits the bioavailability of haem to micro-organisms, but is more active towards the haem-liganded protein.
depreciation in haem scavenging by albumin at periodontal sites through the action of inpa would hinder local haem clearance and potentially advantage p. intermedia and other haem-requiring organisms.
in addition, in regard to the periodontal pocket environment, it is known that in addition to low redox potential, slightly alkaline ph conditions also severely perturb host haem recycling, since they lower the albumin-haem binding affinity  <cit> .
CONCLUSIONS
the current findings show that inpa can function as an albuminase and may enable p. intermedia to specifically target albumin present in whole plasma.
although reducing and slightly alkaline conditions may be optimal for inpa breakdown of albumin and haem release, this protease may also be versatile in degrading albumin in a plaque environment where non-reducing conditions prevail, such as a newly inflamed site or at early stages of periodontal pocket formation.
in conclusion, by more easily degrading haem-liganded albumin, inpa may contribute to haem acquisition by p. intermedia where the availability of this essential growth factor is otherwise tightly restricted by the host.

 members of the genus prevotella are associated with periodontal diseases in humans and other animals. they have a growth requirement for iron protoporphyrin ix, and display a characteristic black-pigmenting phenotype due to cell-surface accumulation of haem protoporphyrin ix) in the monomeric form ppix.oh, haematin), derived via the breakdown of haemoglobin. the haem-containing pigments of both prevotella and porphyromonas species are important defensively, since ferrihaems in both the monomeric and dimeric ppix]2o) forms possess inherent catalase activity and thus can protect against hydrogen peroxide which is produced by activated neutrophils. in the primary habitats of the black-pigmenting anaerobes, the most abundant haem source is haemoglobin. however, in healthy or minimally inflamed gingival crevices, in the absence of overt bleeding, their survival and growth may depend on haemalbumin and haem-haemopexin as primary haem sources. these proteins normally sequester any circulating free haem and transport it to the liver, thus limiting its bioavailability to invading micro-organisms. albumin is the most predominant protein from both healthy and inflamed periodontal disease sites. although albumin has a 75-fold lower affinity for haem compared to haemopexin, its haem sequestering capability is compensated by its 40-80-fold greater concentration in serum, and therefore it binds the majority of any haem liberated into the circulation. in addition, in normal serum, approximately in albumin molecules is haem liganded. importantly, these haemalbumin molecules may serve as a non-haemoglobin source of iron protoporphyrin ix under conditions of haem limitation for p. intermedia and other sub- and supra-gingival plaque bacteria. we have investigated the mechanism of protease-mediated haem release from haemoglobin by p. intermedia. this organism expresses a 90-kda cysteine protease, termed interpain a, an orthologue of periodontain of p. gingivalis and streptopain of streptococcus pyogenes. inpa is likely to serve a major role in pigment production through oxidising oxyhaemoglobin and then fully degrading the methaemoglobin product to release haem. in addition, it has been recently shown that inpa may serve to aid p. gingivalis haem acquisition by proteolytically mediating formation of methaemoglobin from which the p. gingivalis hmuy haemophore can extract haem. cell associated protease activity of p. intermedia can degrade albumin and haemopexin, although the proteases mediating this have not been characterised. incubation of the bacterium with albumin intensifies its proteolytic activity and growth. given that albumin represents an important source of haem under conditions of haem scarcity, we examined the ability of inpa to degrade albumin in both its apo- and haem-liganded forms under conditions which may prevail in the primary habitats of this bacterium, the inflamed gingival sulcus and diseased periodontal pocket.  since the redox potential of diseased periodontal pockets can be as low as − mv and that p. intermedia may experience wide fluctuations of ph within dental plaque, it was considered appropriate to examine the effect of these two variables on the activity of inpa. our previous study showed that inpa activity was stimulated by dithiothreitol and so we examined the effect of various concentrations of dtt on inpa activity versus the fluorescent synthetic substrate z-arg-arg-amc. this revealed that enzyme activity began to plateau at mm. we then determined the influence of the thiol reducing agents cysteine, reduced glutathione and β-mercaptoethanol on inpa activity using the above substrate, and this showed that dtt gave the greatest stimulatory effect. however, in subsequent experiments to examine the influence of reducing conditions on albumin and haemalbumin degradation, we employed mm dtt to ensure full enzyme activation.fig. effect of dithiothreitol and other thiol reducing agents on the enzyme activity of inpa. the enzyme activity was assayed in triplicate using z-arg-arg-amc at °c in m tris–hcl, ph and the concentrations of the reducing agents in fig. 1b were mm. due to the high reproducibility of the replicates, no sd is apparent. control, activity in the absence of reducing agent. inpa concentrations in and were μg ml− see text for details inpa displayed a broad activity against z-arg-arg-amc over the ph range from to. however, for azo-albumin, the maximal activity was centred at ph, with little or no activity beyond ph or above. this may be important in terms of protein breakdown since p. intermedia can generate acid during growth in both liquid and solid media, and may thus depress the ph locally in vivo. we also investigated the temperature activity profile for inpa and thermal stability of the enzyme using azocoll and the synthetic substrate z-arg-arg-amc, respectively. the enzyme showed maximal activity in the temperature range from to °c versus azocoll. however, the enzymatic activity was found to be drastically decreased after the protein was pre-incubated for min at temperatures above °c, followed by assay with z-arg-arg-amc. this indicates that inpa should be highly active at elevated temperatures which may be encountered in vivo in the inflamed gingival sulcus or periodontal pocket during periods of inflammation..fig. effect of ph on inpa activity towards z-arg-arg-amc and azoalbumin. protease activity was assayed at °c in pbs, ph, containing mm dtt. inpa concentration was μg ml− azoalbumin concentration was %. enzyme activity is depicted as absorbance of azo dye released from the protein. see text for details in the absence of dtt, there was little or no degradation of albumin, and densitometry showed that total apoalbumin degradation over h was less than % at all three phs studied. in contrast however, for haemalbumin, three lower molecular weight peptide fragments of rf, and, were generated at ph and. the two fastest migrating of these bands, which stained with tmb-h2o and hence retained bound haem, were further degraded with time and also showed progressive loss of haem staining, as did the main 66-kda haemalbumin band, especially at ph after h. little protein degradation was observed at ph. however, after h, tmb-h2o staining of haemalbumin had decreased by circa % at all phs examined, showing that extensive proteolysis was not necessary to effect haem release from the molecule. in contrast however, the percentage of haemalbumin protein degraded after h was greater at ph, compared to either ph or ph. it should be noted that both apo- and haemalbumin were stable over h at all ph conditions tested in the absence of inpa.fig. albumin and haemalbumin breakdown by inpa at different ph under non-reducing conditions. albumin and haemalbumin were incubated with inpa at °c and aliquots of the incubations were sampled periodically at and h and inpa activity inhibited by e- and subjected to sds-page on % non-reducing gels. samples were solubilised at °c for h in non-reducing application buffer. the haemalbumin gels were stained with tmb to reveal haem-associated peroxidise activity and counterstained with cbb for protein, whilst albumin gels were stained only with cbb. protein loading was ≈  μg per lane. arrows denote digestion fragments carrying bound haem despite inpa displaying optimum activity towards azo-albumin at ph and being more active against aquomethaemoglobin at this ph, it is interesting that haemalbumin degradation by inpa was negligible at ph but greatest at the slightly alkaline ph of. this apparent increase may not reside in inpa activity per se, but may result from structural changes in the albumin molecule occurring in solution at high ph. albumin displays a greater thermal stability at ph than at ph, undergoing a conformational change around ph referred to as the n → b transition, pointing to the b form being more susceptible to inpa, as has been demonstrated for trypsin. this may be physiologically relevant as diseased periodontal pockets and the inflamed gingival sulcus are slightly alkaline. however, sds-page revealed that haem loss also occurred at acid ph despite the low level of proteolysis, suggesting that only limited proteolysis by inpa is required to loosen the haem-albumin association. compared to apoalbumin, haemalbumin is more susceptible to inpa. haem complexing to bovine serum albumin also leads to greater proteolysis by trypsin and chymotrypsin although proteolysis of haemalbumin by whole cells of p. gingivalis is decreased, particularly as the ratio of bound haem to protein increases. nevertheless, conformational changes occurring upon haem ligation may account for inpa attack, enabling inpa to differentiate between apoalbumin and the scarcer haem-liganded form, which represents approximately 1/ of the total albumin molecules in serum under normal physiological conditions. in stark contrast to non-reducing conditions, albumin degradation was greatly increased under reducing conditions at all phs examined, but especially at ph, where complete breakdown was observed after h. the presence of dtt also increased albumin susceptibility at ph. however, at ph less than % was broken down even when dtt was present. albumin was found to be stable under reducing conditions in the absence of inpa as judged by minimal depreciation in cbb staining observed only after h incubation at ph. in contrast to reducing conditions, apoalbumin was less susceptible to inpa, and densitometric scans indicated that approximately % had been degraded after h at each ph.fig. chemical reduction increases the susceptibility of albumin to inpa. albumin was incubated with inpa at different ph at °c under non-reducing and reducing conditions. gel lanes had a nominal loading of μg albumin, and are stained with cbb. samples were prepared and gels run as described for fig. 3 fig. sds-page showing the stability of albumin and haemalbumin under reducing conditions at different ph. albumin and haemalbumin were reduced with mm dtt and incubated at °c. sds-page conditions were the same as those in fig.  the gels were stained with cbb in view the above, and the compositional similarities between gingival crevicular fluid and plasma, it was appropriate to examine the effect of inpa on whole blood plasma, under conditions commonly reflecting those in the inflamed sulcus and periodontal pocket i.e., alkaline ph and low redox potential. sds-page revealed that overnight incubation of whole human plasma with inpa at ph and in the presence of mm dtt, resulted in almost complete breakdown of the albumin band, whilst leaving many other plasma proteins relatively undigested. in comparison, proteinase k, a broad spectrum serine endopeptidase without any peptide bond preference, degraded a large number of the proteins in whole plasma but was less efficient at degrading the albumin component when compared to inpa at the same enzyme concentration. these data show that inpa is able to target albumin present in plasma.fig. sds-page analysis of the breakdown of whole human plasma by inpa. plasma, was incubated overnight at °c in pbs, ph, containing mm dtt in absence or presence of active inpa in a final volume of μl. ~ μg total protein was loaded per lane. arrow denotes albumin band. samples were solubilised for min at °c in reducing application buffer, and gels were stained with cbb fig. comparative sds-page analysis of the digestion of whole human plasma by inpa and proteinase k. whole human plasma was incubated overnight with inpa and proteinase k at the indicated concentrations at °c in pbs, ph containing mm dtt. last lane of each gel was loaded with μg of either inpa or proteinase k only. samples were prepared and gels stained as in fig. 6 the increased susceptibility of albumin to inpa in the presence of dtt may arise from structural changes brought about by chemical reduction of the disulphide bridges. conformational changes of the local tryptophan environment in albumin are reflected in fluctuations of intrinsic fluorescence. when apoalbumin was treated with dtt there was a reduction of fluorescence intensity and moderate shift of λmax, indicating a change in the protein structure. fluorescent emission was not detected above μm dtt, indicating that the concentrations used in the digestions were sufficient to induce structural alterations in albumin. since inpa was pre-activated with an excess of dtt, it is likely that this increased susceptibility of albumin to inpa under reducing conditions was due to structural changes in the substrate rather than to any over-stimulatory effect of dtt on protease activity per se.fig. intrinsic fluorescence spectra of bovine albumin in the presence of dtt. albumin was treated with increasing concentrations of dtt in m nacl, m tris–hcl buffer, ph, at °c, and the fluorescence emission monitored after excitation at nm the low redox potential of diseased periodontal pockets is attributable to the presence of volatile sulphur compounds including h2s and methyl mercaptan, generated by organisms including p. intermedia, p. gingivalis and fusobacterium nucleatum. p. intermedia is also recovered from periodontal disease sites of patients suffering from oral malodour linked to vsc production. the disulphide bridges of albumin are sensitive to reduction, which stimulates tertiary structural changes, leading to increased molecular flexibility and enhanced susceptibility to pepsin. chemical reduction resulting from bacterial metabolism occurs during batch-wise enrichment culture of sub-gingival plaque in serum, where redox potentials of - to − mv have been recorded, and correlate with both h2s production and enrichment in numbers of p. intermedia. since mm dtt was sufficient for optimal inpa activity, and is a concentration which can depress the redox potential to between − and − mv in e. coli growth medium, it is likely that the reducing environment of deep periodontal pockets also results in structural changes in albumin because of disulphide bond disruption, rendering albumin highly susceptible to inpa. predictably therefore, treatment of albumin with mm dtt greatly increased degradation by inpa, especially at ph. the minimal breakdown at acid ph with dtt present, where inpa activity towards azo-albumin and aquomethaemoglobin is highest, suggests that ph-dependent conformational changes may dictate the effectiveness of proteolysis by inpa. it is noteworthy also that accessibility of reducing agents to disulfide bonds in albumin increases above ph. in the presence of mm dtt the susceptibility of haemalbumin to inpa was also dramatically increased. this was especially noticeable at ph; an observation explained by the fact that haemin binding to albumin expands sub-domain ib which may render other regions of the peptide chain available for attack. to further assess the ability of inpa to bring about proteolytic haem release from albumin at ph haemalbumin conjugated agarose was used as a substrate. after h in the presence of dtt, inpa liberated approximately 3-fold more haem than under non-reducing conditions. in this context it is noteworthy that the albumin-haem affinity is lowered under reducing conditions, which in vivo favours haem transfer to haemopexin. furthermore, we observed that under reducing conditions haemalbumin was intrinsically more unstable than its non-liganded counterpart, particularly at alkaline ph, which may explain its heightened susceptibility to enzymic digestion. thus, should the local plaque or periodontal pocket redox potential decline, the additional effect of reducing agents in facilitating haem liberation may supplement inpa-mediated haem release.fig. effect of reducing conditions on breakdown of haemalbumin by inpa at different ph. haemalbumin with a 1: haem to protein ratio was incubated at °c with inpa in the absence and presence of mm dtt. samples and gels were run under non-reducing conditions on % gels and stained for protein with cbb. each gel lane had a nominal loading of μg albumin it is noteworthy that grenier et al. demonstrated albumin breakdown by p. gingivalis r- and k-gingipains in the presence of mm dtt. we have observed that albumin and haemalbumin are resistant to active forms of both hrgpa and kgp under non-reducing conditions. these and the above findings highlight the importance of reducing conditions which prevail in the diseased periodontal pocket and which are essential for disrupting both the haem liganded and apoalbumin structures. inpa has specificity for k, r, a and f residues at the p position of hydrolysed peptide bonds and this was seen in the large number of peptide fragments generated under both reducing and non-reducing conditions. in all, inpa scission sites were exclusive to reducing conditions and to non-reducing conditions, with sites common to digestions under both incubation conditions. the formation of more fragments in the presence of dtt is not surprising, as it may reflect the exposure of more susceptible cleavage sites at previously inaccessible residues resulting from disulphide bond disruption.fig. primary sequence showing all the cleavage sites inflicted during incubation of bovine serum albumin with inpa. albumin was incubated at °c with inpa in mm tris buffer, ph, in the presence or absence of 2mm dtt, and the incubation mixture sampled over time subjected to maldi-tof mass spectrometry. inpa scission sites exclusive to reducing (), non-reducing (), and to both incubation conditions () are indicated. see text for details bleeding, and consequently increased levels of haemoglobin and hence haem, is associated clinically with the inflamed gingival crevice and diseased periodontal pocket, where the host responds to bacterial presence by increasing levels of plasma stress proteins including haemopexin and albumin. in addition, the combined haemolytic and proteolytic activities of other sub-gingival micro-organisms may also transiently elevate local haem levels resulting in greater haem-albumin concentrations. however, we show that inpa not only degrades apoalbumin which normally reduces the potential for haem-induced inflammatory events and limits the bioavailability of haem to micro-organisms, but is more active towards the haem-liganded protein. depreciation in haem scavenging by albumin at periodontal sites through the action of inpa would hinder local haem clearance and potentially advantage p. intermedia and other haem-requiring organisms. in addition, in regard to the periodontal pocket environment, it is known that in addition to low redox potential, slightly alkaline ph conditions also severely perturb host haem recycling, since they lower the albumin-haem binding affinity.  the current findings show that inpa can function as an albuminase and may enable p. intermedia to specifically target albumin present in whole plasma. although reducing and slightly alkaline conditions may be optimal for inpa breakdown of albumin and haem release, this protease may also be versatile in degrading albumin in a plaque environment where non-reducing conditions prevail, such as a newly inflamed site or at early stages of periodontal pocket formation. in conclusion, by more easily degrading haem-liganded albumin, inpa may contribute to haem acquisition by p. intermedia where the availability of this essential growth factor is otherwise tightly restricted by the host. 
inpa may thus contribute significantly to haem acquisition by p. intermedia under conditions of low redox potential and higher ph in the inflamed gingival crevice and diseased periodontal pocket where haem availability is tightly controlled by the host. however, it is not known whether the enzyme may play a role in degrading other haem-carrying plasma proteins present in the gingival sulcus or periodontal pocket from which to derive haem. under these conditions inpa was able to substantially degrade the albumin component of whole human plasma. the bacterium expresses a 90-kda cysteine protease termed interpain a which both oxidizes and subsequently degrades haemoglobin, releasing haem. albumin breakdown was examined over a range of ph and in the presence of reducing agent; conditions which prevail in sub- and supra-gingival plaque.
 prevotella intermedia is a gram-negative black-pigmenting oral anaerobe associated with periodontitis in humans, and has a haem requirement for growth, survival and virulence. it produces an iron porphyrin-containing pigment comprising monomeric iron protoporphyrin ix ppix.oh; haematin). the bacterium expresses a 90-kda cysteine protease termed interpain a which both oxidizes and subsequently degrades haemoglobin, releasing haem. however, it is not known whether the enzyme may play a role in degrading other haem-carrying plasma proteins present in the gingival sulcus or periodontal pocket from which to derive haem. this study evaluated the ability of inpa to degrade apo- and haem-complexed albumin.  albumin breakdown was examined over a range of ph and in the presence of reducing agent; conditions which prevail in sub- and supra-gingival plaque. inpa digested haemalbumin more efficiently than apoalbumin, especially under reducing conditions at ph. under these conditions inpa was able to substantially degrade the albumin component of whole human plasma.  the data point to inpa as an efficient “albuminase” with the ability to degrade the minor fraction of haem-bound albumin in plasma. inpa may thus contribute significantly to haem acquisition by p. intermedia under conditions of low redox potential and higher ph in the inflamed gingival crevice and diseased periodontal pocket where haem availability is tightly controlled by the host. keywords albuminproteaseperiodontal diseasehaeminterpain aprevotella intermediaissue-copyright-statement© the author 2015 