Redox modulation of Rubisco conformation and activity through its cysteine residues

Treatment of purified Rubisco with agents that specifically oxidize cysteine-thiol groups causes catalytic inactivation and increased proteolytic sensitivity of the enzyme. It has been suggested that these redox properties may sustain a mechanism of regulating Rubisco activity and turnover during senescence or stress. Current research efforts are addressing the structural basis of the redox modulation of Rubisco and the identification of critical cysteines. Redox shifts result in Rubisco conformational changes as revealed by the alteration of its proteolytic fragmentation pattern upon oxidation. In particular, the augmented susceptibility of Rubisco to proteases is due to increased exposure of a small loop (between Ser61 and Thr68) when oxidized. Progressive oxidation of Rubisco cysteines using disulphide/thiol mixtures at different ratios have shown that inactivation occurs under milder oxidative conditions than proteolytic sensitization, suggesting the involvement of different critical cysteines. Site-directed mutagenesis of conserved cysteines in the Chlamydomonas reinhardtii Rubisco identified Cys449 and Cys459 among those involved in oxidative inactivation, and Cys172 and Cys192 as the specific target for arsenite. The physiological importance of Rubisco redox regulation is supported by the in vivo response of the cysteine mutants to stress conditions. Substitution of Cys172 caused a pronounced delay in stress-induced Rubisco degradation, while the replacement of the functionally redundant Cys449-Cys459 pair resulted in an enhanced catabolism with a faster high-molecular weight polymerization and translocation to membranes. These results suggest that several cysteines contribute to a sequence of conformational changes that trigger the different stages of Rubisco catabolism under increasing oxidative conditions.     

Cysteines 449 and 459 modulate the reduction-oxidation conformational changes of ribulose 1.5-bisphosphate carboxylase/oxygenase and the translocation of the enzyme to membranes during stress

The role of cysteines 449 (Cys449) and 459 (Cys459) from the large subunit (LS) of ribulose 1-5-bisphosphate carboxylase/oxygenase (Rubisco) in the reduction-oxidation (redox) regulation of the enzyme was assessed by site-directed mutagenesis of these residues and chloroplast transformation of Chlamydomonas reinhardtii. In vitro studies indicated that mutations C449S, C459S or C449S/ C459S do not affect the activity and proteolytic susceptibility of the enzyme in the reduced state. However, when oxidized, the mutant enzymes differed from the wild type (WT), showing an increased resistance to inactivation and, in the case of the double mutant (DM), an altered structural conformation as reflected by the kinetics of proteolysis with subtilisin. The response of the DM strain to saline stress revealed that the absence of Cys449 and Cys459 intensifies Rubisco degradation and the covalent disulfide and non-disulfide polymerization of the enzyme in vivo. Saline stress also induced Rubisco translocation to a membrane (M) fraction that contained only covalently polymerized enzyme. Rubisco mobilization to this M fraction was enhanced also in the DM strain. Altogether, these results indicate that Cys449 and Cys459 participate in the modulation of the conformational changes promoted by oxidative modifications retarding processes related to the catabolism of the enzyme in vivo.     

Increased proteolytic resistance of ribonuclease A by protein engineering.

Although highly stable toward unfolding, native ribonuclease A is known to be cleaved by unspecific proteases in the flexible loop region near Ala20. With the aim to create a protease-resistant ribonuclease A, Ala20 was substituted for Pro by site-directed mutagenesis. The resulting mutant enzyme was nearly identical to the wild-type enzyme in the near-UV and far-UV circular dichroism spectra, in its activity to 2',3'-cCMP and in its thermodynamic stability. However, the proteolytic resistance to proteinase K and subtilisin Carlsberg was extremely increased. Pseudo-first-order rate constants of proteolysis, determined by densitometric analysis of the bands of intact protein in SDS-PAGE, decreased by two orders of magnitude. In contrast, the rate constant of proteolysis with elastase was similar to that of the wild-type enzyme. These differences can be explained by the analysis of the fragments occurring in proteolysis with elastase. Ser21-Ser22 was identified as the main primary cleavage site in the degradation of the mutant enzyme by elastase. Obviously, this bond is not cleavable by proteinase K or subtilisin Carlsberg. The results demonstrate the high potential of a single mutation in protein stabilization to proteolytic degradation.     

Oxidation increases the proteolytic susceptibility of a localized region in rhodanese

For the first time, the enzyme rhodanese has been proteolytically cleaved to give species that most likely correspond to individual domains. This indicates cleavage can occur in the interdomain tether. Further, the conditions for cleavage show that availability of the susceptible bond(s) depends on conformational changes triggered by oxidative inactivation. Rhodanese, without persulfide sulfur (E), was oxidized consequent to incubation with phenylglyoxal, NADH, or hydrogen peroxide. The oxidized enzyme (Eox) was probed using the proteolytic enzymes endoproteinase glutamate C (V8), trypsin, chymotrypsin, or subtilisin. The proteolytic susceptibility of Eox, formed using hydrogen peroxide, was compared with that of E and the form of the enzyme containing transferred sulfur, ES. ES was totally refractory to proteolysis, while E was only clipped to a small extent by trypsin or V8 and not at all by chymotrypsin or subtilisin. Eox was susceptible to proteolysis by all the proteases used, and, although there were some differences among the proteolytic patterns, there was always a band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to Mr = 16,500. This was the only band observed in addition to the parent species (Mr = 33,000) when Eox was digested with chymotrypsin, and conservation of total protein was observed after digestion up to 90 min. No additional species were observable on silver staining, although there was some indication that the band at 16,500 might be a doublet. The results are consistent with the occurrence of a conformational change after oxidation that results in increased exposure and/or flexibility of the interdomain tether which contains residues that meet the specificity requirements of the proteases used.     

Beta-oxidation system of the filamentous fungus Neurospora crassa. Structural characterization of the trifunctional protein.

Treatment of the trifunctional protein from Neurospora crassa with various proteases produced almost identical patterns of proteolytic fragments. To study the structural features of the protein in more detail limited proteolysis with trypsin was carried out. Polyclonal antibodies were raised against three different tryptic fragments. With the help of immunological methods and amino-terminal sequence analysis we were able to monitor the sequential cleavage steps during proteolysis. Two major fragments (an amino-terminal one of 51 kDa and a carboxyl-terminal one of 46 kDa) were identified at the first cleavage step, dividing the 93-kDa subunit of the trifunctional protein almost in half. Additional proteolysis products, deriving from either half, were formed in subsequent proteolytic steps. Combining these results with those obtained from enzyme analysis of the proteolyzed protein, a domain structure of the trifunctional protein is proposed. According to our model each subunit of the tetrameric protein consists of at least two large domains, the amino-terminal one possessing 2-enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase activity and the carboxyl-terminal one bearing 3-hydroxyacyl-CoA epimerase activity.     

Structural characteristics of an abnormal protein influencing its proteolytic susceptibility.

Structural properties of two similar beta-galactosidase fragments were investigated to determine how they influence the fragments' degradation rate in Escherichia coli. Both fragments resulting from a C-terminal nonsense mutation in lacZ, the CSH11 polypeptide and its 90 kDa degradative intermediate, exist predominantly as monomer subunits instead of in the tetrameric form characteristic of the native enzyme. However, both fragments appear to produce trace amounts of dimers and tetramers. The tetramer and higher molecular weight aggregates formed by the wild-type subunit confer greater protection for the enzyme's N-terminal auto-alpha polypeptide than does the monomer state of the beta-galactosidase fragments. The thermally induced aggregation of both beta-galactosidase fragments correlates with their sensitivity to alpha-chymotrypsin. The relatively low thermal stability of the 90 kDa degradative intermediate appears to be the cause of the significant increase in its proteolytic susceptibility at moderately high temperatures.     

Proteolytic modification of rat prolactin by subcellular fractions of the lactating rat mammary gland

The current study explored prolactin proteolysis by rat lactating mammary gland. 125I-labelled rat prolactin was incubated with tissue fractions of lactating mammary gland and the extent of prolactin degradation and fragment formation was visualized and densitometrically quantitated from autoradiographs derived from SDS-polyacrylamide gel electrophoresis under reducing conditions. At pH 4.5, the 25 000 X g pellet of mammary gland converted intact prolactin (23 kDa band) to proteolytic fragments (8-16 kDa bands) in a time- and tissue concentration-dependent fashion similar to that reported previously for rat ventral prostate. The prolactin-degrading and -fragmenting activity in lactating mammary gland was 5-10-times that observed for ventral prostate, the most active male tissue. This activity at acid pH was also demonstrable in other fractions of mammary gland but appeared to predominate in the cytosol. The above activities in mammary gland virtually disappeared at pH 7.4, appeared sensitive to aspartate and sulfhydryl proteinase inhibitors, and insensitive to serine and metalloenzyme proteinase inhibitors. The distribution of this activity could not be correlated with a particular enzyme marker. These characteristics of mammary gland activity differed significantly from those reported previously for prostate. When electrophoresis was conducted under non-reducing conditions, prolactin proteolysis in prostate and mammary gland was primarily associated with the formation of a more slowly migrating product (24 kDa band) with little spontaneous 8-16 kDa fragment formation. Re-electrophoresis of the 24 kDa band under reducing conditions resulted in the appearance of the 8 and 16 kDa fragments. In conclusion, prolactin is proteolytically modified by prostate and lactating mammary gland to a variant of intact hormone (24 kDa band) with a cleavage site in its large loop, by two or more widely distributed, acid-dependent proteinases. Lactating mammary gland, the principal target for prolactin, has the capacity to cleave the hormone in its loop at rates higher than any other tissue examined to date.     

Presence of functional domains in Limulus polyphemus hemocyanin

In an attempt to isolate structural domains of arthropod hemocyanins and possibly to investigate their functional properties, we have undertaken proteolytic digestion experiments of isolated subunits from Panulirus interruptus and Limulus polyphemus oxy-hemocyanin. Satisfactory results have been obtained using trypsin at high concentration and short digestion times. Results show that, in the case of Panulirus hemocyanin, only subunit alpha is susceptible to trypsin digestion, but that proteolytic cleavage is associated with the loss of the copper-oxygen band; on the other hand, in the case of Limulus hemocyanin, four subunits (I, II, III and IV) show a significant susceptibility to trypsin, and their fragmentation takes place with preservation of the oxygen-binding capacity. A more detailed study of the digestion products of subunit IV from Limulus hemocyanin reveals that the proteolytic fragments keep together in a single non-covalent complex. Attempts to separate the native fragments result in the precipitation of the digestion products. Subunit IV of Limulus with proteolytic cuts binds O2 and CO with the same affinity as the native subunit, suggesting that the copper site is still preserved structurally and is functionally active in a 37 kDa trypsin-resistant domain.     

Proteolytic sensitivity of a recombinant phospholipase D from cabbage: identification of loop regions and conformational changes

A recombinant phospholipase D from white cabbage (PLD2) composed of 812 amino acid residues was studied by site-directed mutagenesis and limited proteolysis to obtain first information on its tertiary structure. Limited proteolysis by thermolysin resulted in the formation of some large fragments of PLD2. From mass spectrometry and N-terminal sequencing of the peptides, the cleavage sites could be identified (1. Thr41-Ile42, 2. Asn323-Leu324 or Gly287-Leu288 and Ser319-Ile320 in case of the mutant L324S-PLD2). This suggested an exposed loop in the C2 domain of PLD2 and a large flexible region close to the N-terminal side of the first catalytic (HKD) motif. Calcium ions, the substrate 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and the competitive inhibitor 1,3-dipalmitoylglycero-2-phosphocholine influenced the proteolytic cleavage. Calcium ions exerted a destabilizing effect on the conformation of PLD2.     

The effect of ligand binding on the proteolytic pattern of methylmalonate semialdehyde dehydrogenase

Native rat liver methylmalonate semialdehyde dehydrogenase was proteolyzed by lysylendopeptidase C, chymotrypsin, and trypsin to generate different cleavage fragments of molecular masses: 50, 8, 55, 44, 39, 53, 45, and 40 kDa. A proteolytic cleavage map of MMSDH was constructed based on sequencing data and a comparison of appearance and degradation rates of the different protein fragments as shown by SDS-PAGE. NAD+ was highly effective as a protector against proteolysis in both the N-terminal and the C-terminal parts of the intact enzyme. NADH did not efficiently protect the intact enzyme; however, it stabilized proteolytic fragment L50 from further degradation. This suggests that the NAD(+)-binding domain is not destroyed by cleavage of the N-terminal part of MMSDH. CoA had no effect on the proteolytic cleavage patterns of MMSDH. However, CoA esters reduced the protective effect of NAD+ with an order of effectiveness of acetyl-CoA greater than propionyl-CoA greater than butyryl-CoA. p-Nitrophenyl acetate, substrate for esterase activity by the enzyme, partially prevented the protective effect of NAD+ against proteolysis. These results suggest that S-acylation of the enzyme prevents a stabilizing conformational change induced in MMSDH by NAD+ binding.     

The N-terminal region of the plasma membrane Ca(2+) pump does not separate from the main catalytic fragments after proteolysis

The purified plasma membrane Ca(2+) pump (PMCA) was digested with trypsin, and the proteolytic products were identified by immunoblotting with monoclonal antibodies JA9 or 5F10 directed against the extreme N-terminal segment and the central portion of the molecule, respectively. After a short treatment with low concentrations of the protease, JA9 reacted predominantly with a peptide of 35 kDa whereas 5F10 detected a peptide of 90 kDa. The trypsin cut leading to the production of these fragments had no effect on the maximal activity of the enzyme. At higher concentrations of trypsin, JA9 detected a main fragment of 33 kDa and smaller fragments of 19 and 15 kDa. The persistence of fragments reacting with JA9 indicates that the N-terminal region containing its epitope (residues 51-75) was not easily accessible to the protease in the native PMCA. However, the reactivity with JA9 was rapidly lost during proteolysis of the denatured protein. The passage of the mixture of PMCA fragments through a calmodulin-Sepharose column resulted in the retention of the N-terminal 35 kDa fragment together with that of 90 kDa, despite the fact that only the latter binds calmodulin. The ethylenediaminetetraacetic acid (EDTA) eluate, which contained about equal amounts of both fragments, had a Ca(2+) ATPase activity similar to that of the intact enzyme. The tight association between the two peptides was evidenced by the fact that concentrations of polyoxyethylene 10 lauryl ether (C(12)E(10)), sodium dodecyl sulfate (SDS) high enough for inactivating the enzyme and dissociate the pump from calmodulin were unable of breaking the interaction between the 35 and 90 kDa fragments. Altogether, these results show that after digestion with trypsin, the N-terminal portion of the PMCA, including the extreme N-terminal segment, remains part of a fully functional catalytic complex.     

Cloning, sequence analysis and over-expression of the gene for the class II fructose 1,6-bisphosphate aldolase of Escherichia coli.

An early event in malignant transformation is the increased expression of proteases, such as plasminogen activator, which can degrade surrounding extracellular matrices, thereby conferring an advantage for tumour cell invasion and metastasis. The present studies provide evidence that plasma fibronectin (Fn), which is a component of the extracellular matrix, is a direct substrate for the plasminogen activator urokinase (UK). Human plasma Fn was incubated with human UK under plasminogen-free conditions. Fn cleavage was both time- and dose-dependent and was evident within 30 min. The proteolytic digestion was limited and complete within 12 h at an enzyme/substrate ratio of 1:20. Analysis of the final proteolytic digestion products demonstrated the disappearance of the native dimeric 440 kDa structure of Fn with the concomitant appearance of three proteolytic fragments of 210, 200 and 25 kDa. Since two large fragments of similar size to the 220 kDa monomeric chains of Fn were obtained following proteolysis, it is proposed that UK cleaves Fn at two sites, one towards the N-terminal and one close to the C-terminal, but N-terminal to its interchain disulphide bonds. These studies suggest that the local proteolytic digestion and release of Fn from the extracellular matrix by tumour cells possessing high levels of UK may involve the direct proteolytic breakdown of Fn by UK.     

Structural aspects of myosin subfragment 1 as studied by subtilisin digestion of trypsin-split S-1 (27 kDa-50 kDa-20 kDa)

Limited subtilisin digestion of myosin subfragment 1 (S-1) was carried out, varying the enzyme: substrate weight ratio from 1:200 to 1:10, and changes in structure, and in the MgATPase activities of S-1 and acto-S-1 after proteolysis, were followed. When the starting material--tryptically-cleaved S-1 (27 kDa-50 kDa-20 kDa) ("split S-1")--was subjected to further subtilisin digestion, it was found that with increasing enzyme concentration, the 50 kDa fragment degraded into an 18 kDa fragment via a 33 kDa peptide (50----33----18 kDa), which was not cross-linked with F-actin. On the other hand, the 27 and 20 kDa fragments were rather stable at lower subtilisin concentrations and started to degrade only at higher subtilisin concentrations. These degradations lowered the MgATPase activities of S-1 and acto-S-1. The losses of MgATPase activities of S-1 and of acto-S-1 were mainly due to the degradations of the 27 and 20 kDa fragments, respectively. Addition of EDTA did not affect the subtilisin cleavage pattern of split S-1 but the breakdown of the 50 kDa fragment was extremely depressed, suggesting that some conformational change of the 50 kDa fragment is induced by the binding of divalent cation. The binding of MgADP to split S-1 accelerated the degradation of the 27 kDa fragment and produced a new cut in the 27 kDa fragment (27----20 kDa), resulting in a further loss of the S-1 MgATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Limited proteolysis of pig liver CoA synthase: evidence for subunit identity

The bifunctional enzyme CoA synthase can be nicked by trypsin without loss of its activities. The original dimer of subunit Mr approx. 61 000 yields fragments of Mr 41 000 and 22 000 as seen on gel electrophoresis in the presence of SDS, but the nicked enzyme retains the native Mr of 118 000. Further proteolysis occurs rapidly in the absence of protecting substrates. The N-terminal of native CoA synthase is proline, and proteolysis exposes glycine as a second N-terminal. This evidence strongly suggests that the subunits are identical.     

Refolding kinetics of pig muscle and yeast 3-phosphoglycerate kinases and of their proteolytic fragments.

The time course of refolding of both pig muscle and yeast 3-phosphoglycerate kinase (molecular masses about 47 kDa), as well as their proteolytic C-terminal fragments (30 and 33 kDa, respectively) has been investigated. Very similar refolding kinetics (with half-time between 80-120 s, at 20 degrees C) were observed by fluorescence and ultraviolet absorbance spectroscopy, as well as by activity measurements, for the intact enzyme from both sources. This time course appears not to depend on the time the protein spends in the unfolded state, i.e. it is certainly not controlled by proline isomerization. Furthermore, after removal of a large N-terminal part (molecular mass of about 18 kDa for pig muscle enzyme or 13 kDa for yeast enzyme) of the molecule by proteolysis, refolding of the remaining C-terminal fragment of both proteins follows kinetics virtually indistinguishable from those of the intact protein molecule.     

Limited proteolysis as a structural probe of the soluble alpha-glycerophosphate oxidase from Streptococcus sp

As reported previously [Parsonage, D., Luba, J., Mallett, T. C., and Claiborne, A. (1998) J. Biol. Chem. 273, 23812-23822], the flavoprotein alpha-glycerophosphate oxidases (GlpOs) from a number of enterococcal and streptococcal sources contain a conserved 50-52 residue insert that is completely absent in the homologous alpha-glycerophosphate dehydrogenases. On limited proteolysis with trypsin, the GlpO from Streptococcus sp. (m = 67.6 kDa) is readily converted to two major fragments corresponding to masses of approximately 40 and 23 kDa. The combined application of sequence and mass spectrometric analyses demonstrates that the 40-kDa fragment represents the N-terminus of intact GlpO (Met1-Lys368; 40.5 kDa), while the 23-kDa band represents a C-terminal fragment (Ala405-Lys607; 22.9 kDa). Hence, limited proteolysis in effect excises most of the GlpO insert (Ser355-Lys404), indicating that this represents a flexible region on the protein surface. The active-site and other spectroscopic properties of the enzyme, including both flavin and tryptophan fluorescence spectra, titration behavior with both dithionite and sulfite, and preferential binding of the anionic form of the oxidized flavin, were largely unaffected by proteolysis. Enzyme-monitored turnover analyses of the intact and nicked streptococcal GlpOs (at [GlpO] approximately 10 microM) demonstrate that the single major catalytic defect in the nicked enzyme corresponds to a 20-fold increase in K(m)(Glp); the basis for this altered kinetic behavior is derived from an 8-fold decrease in the second-order rate constant for reduction of the nicked enzyme, as measured in anaerobic stopped-flow experiments. These results indicate that the flexible surface region represented by elements of the GlpO insert plays an important role in mediating efficient flavin reduction.     

Interacting domains in the epithelial sodium channel that mediate proteolytic activation

Epithelial Sodium Channel (ENaC) proteolysis at sites in the extracellular loop of the α and γ subunits leads to marked activation. The mechanism of this effect remains debated, as well as the role of the N- and C-terminal fragments of these subunits created by cleavage. We introduced cysteines at sites bracketing upstream and downstream the cleavage regions in α and γ ENaC to examine the role of these fragments in the activated channel. Using thiol modifying reagents, as well as examining the effects of cleavage by exogenous proteases we constructed a functional model that determines the potential interactions of the termini near the cleavage regions. We report that the N-terminal fragments of both α and γ ENaC interact with the channel complex; with interactions between the N-terminal γ and the C-terminal α fragments being the most critical to channel function and activation by exogenous cleavage by subtilisin. Positive charge modification at a.a.135 in the N-terminal fragment of γ exhibited the largest inhibition of channel function. This region was found to interact with the C-terminal α fragment between a.a. 205 and 221; a tract which was previously identified to be the site of subtilisin's action. These data provide the first evidence for the functional channel rearrangement caused by proteolysis of the α and γ subunit and indicate that the untethered N-terminal fragments of these subunits interact with the channel complex.     

Structural domains in the type III restriction endonuclease EcoP15I: characterization by limited proteolysis, mass spectrometry and insertional mutagenesis

The Type III restriction endonuclease EcoP15I forms a hetero-oligomeric enzyme complex that consists of two modification (Mod) subunits and two restriction (Res) subunits. Structural data on Type III restriction enzymes in general are lacking because of their remarkable size of more than 400 kDa and the laborious and low-yield protein purification procedures. We took advantage of the EcoP15I-overexpressing vector pQEP15 and affinity chromatography to generate a quantity of EcoP15I high enough for comprehensive proteolytic digestion studies and analyses of the proteolytic fragments by mass spectrometry. We show here that in the presence of specific DNA the entire Mod subunit is protected from trypsin digestion, whereas in the absence of DNA stable protein domains of the Mod subunit were not detected. In contrast, the Res subunit is comprised of two trypsin-resistant domains of approximately 77-79 kDa and 27-29 kDa, respectively. The cofactor ATP and the presence of DNA, either specific or unspecific, are important stabilizers of the Res subunit. The large N-terminal domain of Res contains numerous functional motifs that are predicted to be involved in ATP-binding and hydrolysis and/or DNA translocation. The C-terminal small domain harbours the catalytic center. Based on our data, we conclude that both structural Res domains are connected by a flexible linker region that spans 23 amino acid residues. To confirm this conclusion, we have investigated several EcoP15I enzyme mutants obtained by insertion mutagenesis in and around the predicted linker region within the Res subunit. All mutants tolerated the genetic manipulation and did not display loss of function or alteration of the DNA cleavage position.     

Purification of human erythrocyte proteolytic enzyme responsible for degradation of oxidant-damaged hemoglobin. Evidence for identifying as a member of the multicatalytic proteinase family

Exposure of human red cells to oxidants such as phenylhydrazine, 2,4-dimethylphenylhydrazine and 4-hydrazinobenzoic acid stimulates the proteolysis of hemoglobin as evidenced by the increase in the rate of the free alanine and acid soluble amino groups released. An enzyme responsible for proteolytic degradation of oxidized hemoglobin, was purified from cytosolic fraction of erythrocytes by a DEAE-batch procedure followed by gel-filtration and ion-exchange chromatography. The final enzyme preparation produces a single band in non-denaturing polyacrylamide gel electrophoresis, and eight different bands of 23-32 kDa when subjected to polyacrylamide gel electrophoresis under denaturing conditions. The native enzyme has a molecular mass of about 700 kDa as estimated by gel filtration. The enzyme, unable to hydrolyze native hemoglobin, cleaves phenylhydrazine-treated hemoglobin into small peptides without free amino acid release. In addition, the enzyme shows an endopeptidase activity towards synthetic peptides having a tyrosine or an arginine in the P1 position, whereas it does not hydrolyze shorter peptides and those with a proline in the P1 or P2 position. The proteolytic activity of the enzyme against oxidized hemoglobin is inhibited by chymostatin and p-chloromercuribenzoate, while it is stimulated by N-ethylmaleimide and epoxysuccinylleucylamido-(4-guanidino)butane (E-64). The peptidase activity assayed on succinyl-Leu-Leu-Val-Tyr-MCA is inhibited by chymostatin, hemin, N-ethylmaleimide and p-chloromercuribenzoate. The results obtained show that in human erythrocytes oxidized hemoglobin is cleaved into peptides by a high molecular mass proteinase identified as a member of the multicatalytic proteinase family. It is also suggested that the complete degradation of oxidized hemoglobin to free amino acids requires the involvement of a further proteolytic enzyme(s) which remain(s) to be identified.