peptide inhibitor of complement c1 inhibits the peroxidase...

Research ArticlePeptide Inhibitor of Complement C1 Inhibits the PeroxidaseActivity of Hemoglobin and Myoglobin

Pamela S Hair1 Kenji M Cunnion1234 and Neel K Krishna12

1Department of Pediatrics Eastern Virginia Medical School 700 West Olney Road Norfolk VA 23507 USA2Department of Microbiology and Molecular Cell Biology Eastern Virginia Medical School 700 West Olney RoadNorfolk VA 23507 USA3Childrenrsquos Specialty Group 811 Redgate Avenue Norfolk VA 23507 USA4Childrenrsquos Hospital of The Kingrsquos Daughters 601 Childrenrsquos Lane Norfolk VA 23507 USA

Correspondence should be addressed to Neel K Krishna krishnnkevmsedu

Received 12 April 2017 Revised 6 July 2017 Accepted 3 August 2017 Published 10 September 2017

Academic Editor Tzi Bun Ng

Copyright copy 2017 Pamela S Hair et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Hemoglobin is the natural carrier of oxygen in red blood cells (RBCs) While intracellular hemoglobin provides life-sustainingoxygen transport extracellular free hemoglobin displays toxicity due to inherent peroxidase activity generating reactive oxygenspecies that subsequently react with the hemoglobinmolecule to produce toxic heme degradation products resulting in free radicalsoxidative stress damage and lipid peroxidation We have recently demonstrated that Peptide Inhibitor of Complement C1 (PIC1)inhibits peroxidase activity of the heme-based enzymemyeloperoxidase To elucidatewhether PIC1 could inhibit peroxidase activityof hemoglobin we evaluated the consequence of PIC1 on RBC lysates methemoglobin andmyoglobin using tetramethylbenzidine(TMB) as an oxidation target PIC1 reversibly and dose-dependently prevented TMB oxidation to tetramethylbenzidine diimineby RBC lysates methemoglobin and myoglobin having comparable activity to the inhibitor 4-aminobenzoic acid hydrazide PIC1inhibited TMB oxidation of RBC lysates similar to L-cysteine suggesting that the two cysteine residues contained in PIC1 maymediate peroxidase activity PIC1 also inhibited heme destruction byNaOCl for RBC lysates hemoglobin andmyoglobin as assayedby preservation of the Soret absorbance peak in the presence of NaOCl and reduction in free iron release In conclusion PIC1inhibits peroxidase activity of hemoglobin and myoglobin likely via an antioxidant mechanism

1 Introduction

The hemoglobin (Hb) molecule is a protein tetramer com-posed of two alpha and two beta chains each of whichcoordinates an iron-containing heme prosthetic group [1]Hb constitutes up to 92 of the dry content of the redblood cell (RBC) and plays the essential role of deliveringoxygen from the lung to the peripheral tissue for cellularuptake reciprocally removing carbon dioxide from the tissueto the lung [1] While Hb provides life-sustaining oxygendelivery the Hb molecule is inherently toxic due to itswell-characterized redox activity leading to autoxidation ofthe ferrousHb (Fe2+) oxygen containing Hb (oxyHb) intononfunctional ferricHb (Fe3+) also known asmethemoglobin(metHb) and reactive oxygen species (ROS) generation ofsuperoxide ion (O

2

∙minus) [2] Within the confines of the RBC

autoxidation is minimized by the metHb reductase systemhowever free metHb can undergo a further peroxidasereaction with O

2

∙minus to generate ferrylHb (Fe4+) and the ROShydrogen peroxide (H

2O2) that in turn attacksHb to produce

protein radicals irreversibly damaging the Hb moleculeleading to heme degradation and release of free iron [2]In addition to O

2

∙minus and H2O2 the ROS hypochlorous acid

(HOCl) generated by myeloperoxidase (MPO) present inneutrophil andmacrophage granules has been demonstratedto bind to the heme moiety of Hb causing heme destructionprotein modification and protein aggregation as well asformation of additional peroxidase-like activity [3 4]

Hb peroxidase activity and its degradation are extremelytoxic to organs and tissues having serious pathologicalramifications [5 6] In addition to generation of ROS fromfree Hb iron released from heme degradation can result in

HindawiInternational Journal of PeptidesVolume 2017 Article ID 9454583 10 pageshttpsdoiorg10115520179454583

2 International Journal of Peptides

the generation of additional ROS resulting in cellular damageFurther reaction of free iron with H

2O2forms a potent

oxidant that is damaging to lipids nucleic acids and aminoacids Finally Hb protein radical formation can result incrosslinking of heme and amino acids [2] Currently there areno pharmacological agents available on the market to controlpathological extracellular Hb-mediated oxidative reactions

Our laboratory has characterized a family of peptidesknow as Peptide Inhibitor of Complement C1 (PIC1) thatinhibit the classical pathway of complement by bindingthe initiator molecule C1q and inhibiting activation of thecognate serine protease tetramer C1s-C1r-C1r-C1s that isnormally associatedwith C1q [7 8]We have recently demon-strated that the lead pegylated PIC1 compound PA-dPEG24(amino acid sequence IALILEPICCQERAA containing a 24units PEGmoiety attached to the C terminus total molecularweight 2771Da) can inhibit MPO activity in cystic fibrosissputum as well as from neutrophils and purified MPO [9]To ascertain if PIC1 had any inhibitory activity on Hb whichshares with MPO a heme-based mechanism of action weanalyzed the effect of PIC1 on the peroxidase activity of Hbfrom RBCs PIC1 was able to inhibit this activity by Hb aswell as purified metHb and myoglobin and also protect itsdestruction by sodiumhypochlorite (NaOCl) suggesting thatPIC1 possesses a novel antioxidant activity that may be oftherapeutic benefit

2 Materials and Methods

21 Ethics Statement Human blood was obtained from fourhealthy volunteers for the generation of RBC lysates usedin these studies per the Eastern Virginia Medical SchoolIRB approved protocol 02-06-EX-0216 The study partic-ipants provided written informed consent Human bloodwas collected for these experiments from May 2016 throughNovember 2016

22 Materials PIC1 (IALILEPICCQERAA-dPEG24) wasmanufactured by PolyPeptide Group (San Diego CA) toge95 purity as verified by HPLC and mass spectrometryanalysis Lyophilized PIC1 was solubilized in normal salinewith 001M Na

2HPO4buffer to 15mM dPEG24 was pur-

chased from PolyPeptide Group and dissolved in the samebuffer Pure human methemoglobin (metHb) myoglobinfrom equine skeletal muscle and L-cysteine were purchasedfrom Sigma (St Louis MO) 331015840551015840-Tetramethylbenzidine(TMB) as a solution containing H

2O2was purchased from

Thermo Fisher Scientific (Waltham MA) 4-Aminobenzoicacid hydrazide (ABAH) O-dianisidine and NaOCl werepurchased from Sigma-Aldrich (St Louis MO) 221015840-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) solu-tion was purchased fromThermo Fisher Scientific (WalthamMA)

23 Preparation of Human RBC Lysate RBC lysates wereprepared from the blood of healthy donors as previouslydescribed [10] Cells were washed twice with GVBSminusminus(veronal-buffered saline 01 gelatin and 001M EDTA) andthen incubated at 37∘C to allow for spontaneous lysis The

cells were then washed twice again with GVBS++ (veronal-buffered saline 015mM CaCl

2 and 10mM MgCl

2) and

resuspended in a volume which set the concentration to 1times 109 cellsml (absorbance reading of lysed RBCs at 541 nmof 0210 = 1 times 109 cellsml) An aliquot of these cells wascombinedwith an equal aliquot of water at room temperatureand spun at 21000timesg for 5 minutes to separate the releasedhemoglobin from the cell debris

24 Measurement of Peroxidase Inhibition by PIC1 Inhibi-tion of peroxidase activity was assessed by combining theRBC lysate (reported in cellular equivalentsml) metHb(05mgml) or myoglobin (05mgml) with increasing con-centrations of PIC1 with a constant final volume for 5minutes before adding TMB as described below Additionallyincreasing amounts of RBC lysates were incubated withincreasing amounts of PIC1 with a constant final volume for5 minutes before adding TMB As a positive control for inhi-bition of peroxidase activity increasing amounts of ABAHor L-cysteine were included in select experiments After the5-minute incubation samples were combined with 100120583l ofTMB in a nontreated 96-well plate for 15 seconds followedby 100 120583l of 25N H

2SO4(stop buffer) and absorbance was

read at 450 nm in a Synergy HT BioTek plate reader [10] Forassays usingABTS 150 120583l of ABTS solutionwas added to eachsample and incubated for 15 seconds before adding 100120583lof 1 SDS to stop the reaction [11] Absorbance was readat 405 nm O-Dianisidine working solution was prepared bymaking a 0166mgml solution in 50mM phosphate-citratebuffer pH 50with a final of 0006H

2O2added immediately

before use [12] 100 120583l of the prepared O-dianisidine solutionwas added to each sample and incubated for 2 minutes beforeadding 100 120583l of 25NH

2SO4to stop the reactionAbsorbance

was read at 405 nmTo assess if PIC1 could inhibit peroxidase activity of Hb

released from hemolyzed RBCs in human serum type Osera at 15 in GVBS++ buffer was mixed with type ABRBCs at titrating concentrations (1 2 dilutions of 5 times 107RBCs) followed by incubation for 1 hour at 37∘C to allow forRBC hemolysis via antibody mediated complement lysis [13]Samples were then centrifuged and supernatant containingthe released Hb collected PIC1 at 1mM final concentrationwas subsequently combined with the sample supernatantsand allowed to incubate for 5 minutes before adding TMBand H

2SO4as above to evaluate peroxidase activity from the

cell-free Hb

25 Reversal of Peroxidase Activity by PIC1 RBC lysate(25 times 108 cellsml equivalent) 2mgml metHb or 2mgmlmyoglobin was coated onto an Immunlon-2 96-well plate ina carbonate coating buffer (016M Na

2CO3 034 NaHCO

3

pH 96) overnight at 4∘C The plate was then washed TMBwas added and allowed to incubate for 1 hour The oxidizedTMBwas transferred to a freshwell containing eitherwater or2mM PIC1 The reaction was stopped after 1min with 25MH2SO4 Absorbance was read at 450 nm

26 Free IronAssay Iron release from theRBC lysatemetHbor myoglobin was measured utilizing a ferrozine assay [14]

International Journal of Peptides 3

After the absorbance spectra were recorded 100 120583l of ascorbicacid (100mM)was added to each sample and allowed to incu-bate for 5 minutes Then 50 120583l of ammonium acetate (16)and 50 120583l of ferrozine (16mM) were added to the samplesand mixed After incubating for 5 minutes absorbance wasmeasured at 562 nm A standard curve was prepared usingammonium Fe(III) sulfate and a linear regression was usedto calculate the free iron concentration

27 Spectral Analysis of RBC Lysate and Pure metHbAbsorbance spectra experiments were performed with 125times 107 cellsml RBC lysate 02mgml metHb or 02mgmlmyoglobin followed by various concentrations of PIC1ABAH and NaOCl (at a 1 1000 dilution) in a nontreated96-well plate As each component was added to the RBClysate metHb or myoglobin it was allowed to incubate for5 minutes Absorbance values were recorded from 300 to550 nm [14]

28 Statistical Analysis Quantitative data were analyzeddetermining means standard error (SEM) and Studentrsquos119905-test using Excel (Microsoft CA) 119875 values le 005 wereconsidered statistically significant

3 Results

31 PIC1 Inhibits Peroxidase Activity of Hb in RBC Lysates Inan attempt to ascertainwhether PIC1would have any effect onthe peroxidase activity of extracellular Hb RBC lysates wereprepared from four donors and incubated with increasingamounts of PIC1 followed by TMB as the target for oxidationAddition of PIC1 led to a dose-dependent inhibition ofthe TMB oxidation product 331015840551015840-tetramethylbenzidinediimine (Figure 1(a)) demonstrating a 248-fold reduction inperoxidase activity for 75mM PIC1 compared with no PIC1(119875 lt 0001) In order to further evaluate PIC1 inhibition ofperoxidase activity over a range of RBC lysate concentrationsa more extensive dose-response experiment was conductedwith RBC lysates from one donor Concentration-dependentPIC1 inhibition of RBC lysate peroxidase activity was demon-strated for all RBC lysate concentrations (Figure 1(b)) PIC1at 375mM inhibited peroxidase activity 143-fold comparedwith no PIC1 at 125 times 108 RBCml equivalents (119875 = 0007)

In an attempt to assess the peroxidase activity of PIC1 ina scenario mimicking intravascular hemolysis the followingexperiment was performed Increasing amounts of humanAB RBCs were incubated in the presence of O serum whichcontains antibodies to the A and B glycoproteins resultingin complement-mediated lysis of the RBCs [13] When PIC1was added to the serum sample containing the lysed RBCsfollowed by TMB PIC1 was found to inhibit peroxidaseactivity of the released Hb compared to the no PIC1 control24-fold at 50 times 107 RBCs (Figure 1(c)) These results suggestthat PIC1 can inhibit peroxidase activity of Hb from RBCslysed in a complement-mediated reaction in human serum

32 PIC1 Reversibly Inhibits Peroxidase Activity of RBCLysates metHb and Myoglobin in a Comparable Manner toABAH To ascertain that the PIC1-mediated inhibition of

peroxidase activity demonstrated for the RBC lysate wasindeed due to Hb alone PIC1 was incubated with puremetHb As a positive control for peroxidase inhibition 4-aminobenzoic acid hydrazide (ABAH) was utilized ABAHis an inhibitor that specifically and irreversibly inhibits theperoxidase activity of MPO a heme containing enzymeproduced by neutrophils and macrophages [15 16] On amolar basis PIC1 demonstrated a similar dose-dependentinhibition of the peroxidase activity of metHb as seen withABAH (Figure 2(a)) At 125mM PIC1 exhibited a 64-fold(119875 = 0004) improvement in metHb inhibition comparedwith ABAH As expected the PIC1 inhibition of metHbperoxidase activity was very similar to that of the RBC lysatetested in parallel which demonstrated superior activity toABAH (Figure 2(b)) At 125mM PIC1 showed a 56-fold(119875 = 0009) improvement in Hb inhibition compared withABAH In order to determine if the peroxidase inhibitoryactivity of PIC1was active against another toxic heme-bearingenzyme the TMB assay was performed with puremyoglobinPIC1 dose-dependently inhibited the peroxidase activity ofmyoglobin by 143-fold (119875 = 0006) at 5mM comparedwith no PIC1 (Figure 2(c)) PIC1 inhibition of myoglobinperoxidase activity was not statistically different fromABAHPolyethylene glycol (PEG) has been previously reported tohave antioxidant capacity [17] To ascertain whether theobserved peroxidase inhibiting activity of PIC1 was due to itsC terminal PEG tail which is composed of 24 polyethyleneglycol units (PEG24) PIC1 or purified PEG24 was incubatedwith metHb While PIC1 dose-dependently inhibited peroxi-dase activity PEG24 had no effect (Figure 2(d))

To rule out that PIC1 inhibition of extracellular Hb-mediated TMB oxidation in this assay was specific to thesubstrate used the reagents ABTS and O-dianisidine wereutilized in place of TMB Both PIC1 and ABAH showeddose-dependent inhibition of free Hb peroxidase activityfor ABTS (Figure 3(a)) and O-dianisidine (Figure 3(b)) [1112] Additionally PIC1 incubated in the presence of TMBalone did not result in a change in absorbance at 450 nmconfirming that PIC1 does not directly reduce the reporterprobe Collectively these data suggest that PIC1 is able tobroadly inhibit the peroxidase activity of the heme-bearingenzymes Hb and myoglobin

PIC1 contains vicinal cysteine residues at positions 9 and10 of the peptide It is well established that the thiol groupof cysteine residues are efficient peroxidase inhibitors with L-cysteine demonstrating significant antioxidant activity [18]In order to evaluate the level of PIC1 peroxidase inhibitioncompared with the cysteine equivalent molar amounts of L-cysteine and PIC1 were added to RBC lysates and analyzedin the TMB assay PIC1 and L-cysteine both demonstrated adose-dependent and similar inhibition of peroxidase activity(Figure 4(a)) This data suggests that the vicinal cysteines ofPIC1 could mediate the peroxidase inhibitory activity of thepeptide

In an attempt to ascertain whether PIC1 could beinhibiting peroxidase activity via an antioxidant effect (iedetermine whether oxidation can be reversed) the followingexperimentwas conducted RBC lysatemetHb ormyoglobinwas coated onto a 96-well plate and incubated overnightThe


PIC1 (mM)0 1 2 3 4 5 6 7 8

00

05

10

15

20

25

30

35

40

Donor 1Donor 2

Donor 3Donor 4

Abs (

450Ｈ

m)

(a)

PIC1 (mM)00 05 10 15 20 25 30 35 40

00

05

10

15

20

25

30

35

40

RBC lysate (125 times 108 cellsml eq)





Abs (

450Ｈ

m)

(b)

0 1 2 3 4 500

05

10

15

20

25

30

35

No PIC11ＧM PIC1

RBC (times107)

Abs (

412Ｈ

m)

(c)

Figure 1 PIC1 inhibits peroxidase activity of Hb in RBC lysates (a) RBC lysates (125 times 108 cellsml equivalents (eq)) from four donorswere incubated with increasing concentrations of PIC1 After a 5-minute incubation samples were combined with TMB for 15 seconds andabsorbance was read at 450 nm Data are means of three independent experiments plusmn SEM (b) Dose-response inhibition of RBC lysates fromone donor at various concentrations by PIC1 Data are means of four independent experiments plusmn SEM (c) Increasing amounts of human ABRBCswere added to humanO serumand cells allowed to lyse PIC1was then added to the serumaliquots and after a 5-minute incubation sam-ples were combined with TMB for 15 seconds and absorbance was read at 412 nm Data are means of three independent experiments plusmn SEM

plate was then washed and TMB added for 1 hour to allowthe substrate to oxidize Each solution was then transferredto a fresh well which contained either water or PIC1 PIC1was able to reverse the oxidation state of TMB for the RBClysate metHb and myoglobin as measured by absorbance at450 nm with a 72ndash84-fold (119875 le 0003) reduction in oxidizedTMB (331015840551015840-tetramethylbenzidine diimine) for each of theproteins incubated with PIC1 compared to the water onlycontrol (Figure 4(b))

33 PIC1 Prevents Hb and Myoglobin Destruction and IronRelease Mediated by NaOCl Hypochlorous acid (HOCl)

produced by MPO present in phagocyte granules has beenshown to mediate heme destruction and free iron release[3 4] HOCl induced changes to the heme ring and iron atomof Hb may be evaluated by measuring spectral absorption tomonitor the Soret peak which is characteristic of porphyrincontaining compounds such as the heme moiety of Hb andmyoglobin [4] To ascertain if PIC1 was able to protect theheme group from hypochlorite attack RBC lysates metHband myoglobin were incubated with increasing concentra-tions of PIC1 followed by NaOCl and spectral analysis per-formedCompared to noPIC1 treatment (NaOCl alone) RBClysates incubated with increasing amounts of PIC1 showed a


Inhibitor (mM)0 1 2 3 4 5

00

04

08

12

16

20

PIC1ABAH

Abs (

450Ｈ

m)

(a)


00

05

10

15

20

25

30

35

PIC1ABAH

Abs (

450Ｈ

m)

(b)


00

02

04

06

08

10

PIC1ABAH

Abs (

450Ｈ

m)

(c)

Inhibitor (mgml)0 5 10 15 20

00

05

10

15

20

PIC1PEG24

Abs (

450Ｈ

m)

(d)

Figure 2 Comparison of PIC1 and ABAH inhibition of peroxidase activity of metHb Hb from RBC lysates and myoglobin (a) 05mgmlmetHb (b) RBC lysates (125 times 108 cellsml equivalents) or (c) 05mgml myoglobin were incubated with increasing concentrations of PIC1and ABAH Samples were then combined with TMB and absorbance was read at 450 nm Data are means of three independent experimentsplusmn SEM (d) 05mgml metHb was incubated with increasing concentrations of PIC1 and PEG24 Samples were then combined with TMB andabsorbance was read at 450 nm

dose-dependent protection of the Soret peak (Figure 5(a))The same result was obtained for pure metHb (Figure 5(b))andmyoglobin (Figure 5(c)) To assess whether PIC1 also pre-vented the release of free iron from the heme group ofHb andmyoglobin the ferrozine assay was performed on these sam-ples PIC1 dose-dependently inhibited free iron release forRBC lysates metHb and myoglobin (119875 le 0003 for 25mMPIC1 versus no PIC1) (Figure 5(d)) consistent with the spec-tral absorbance data showing protection of the heme group

Given the similar levels of peroxidase inhibition byABAH and PIC1 shown in the TMB assay (Figures 2(a)ndash2(c))we next analyzed whether ABAH produced the same protec-tive effect against NaOCl as seen with PIC1 ABAH showedprotection of the Soret peak of Hb from the RBC lysate(Figure 6(a)) as well as metHb (Figure 6(b)) and myoglobin(Figure 6(c)) Interestingly in all three cases ABAH did

not demonstrate a dose-dependent inhibition as seen withPIC1 each concentration of ABAH protected the Soretpeak of Hb to a similar degree Additionally while variousconcentrations of ABAH prevented flattening of the Soretpeak in the presence of NaOCl this inhibitor was unable toblock release of free iron in a dose-dependent fashion for theRBC lysate metHb and myoglobin (Figure 6(d)) These datasuggest that PIC1 protection of these enzymes from NaOClmay occur through a differentmechanismof action thanwhatis observed for ABAH

4 Discussion

Heme is an essential component of proteins such as Hband myoglobin where it functions in life-sustaining oxygentransport and storage respectively However severe hemol-ysis leading to release of free Hb plays a significant role in


PIC1ABAH


00

05

10

15

20

25

30Ab

s (405Ｈ

m)

(a)

PIC1ABAH


00

02

04

06

08

10

12

14

Abs (

405Ｈ

m)

(b)

Figure 3 Comparison of PIC1 and ABAH inhibition of peroxidase activity of Hb from RBC lysates using ABTS and O-dianisidine assubstrates RBC lysates (125 times 108 cellsml equivalents) were incubated with increasing concentrations of PIC1 or ABAH Samples werethen combined with (a) ABTS or (b) O-dianisidine and absorbance was read at 405 nm Data are means of three independent experiments plusmnSEM

Inhibitor (mM)0 1 2 3 4 5 6 7 8

00

05

10

15

20

25

30

35

40

CysteinePIC1

Abs (

450Ｈ

m)

(a)

00

01

02

03

04

05

RBC lysateMethemeMyoglobin

Wat

er

Wat

er

Wat

er

PIC1

PIC1

PIC1

Abso

rban

ce (4

50Ｈ

m)

(b)

Figure 4 PIC1 and cysteine show similar inhibition of TMB oxidation that is reversible (a) RBC lysates were incubated with increasingconcentrations of PIC1 and L-cysteine Samples were then combined with TMB and absorbance was read at 450 nm Data are means of threeindependent experiments plusmn SEM (b) RBC lysate (25 times 108 cellsml equivalents) 2mgml metHb or 2mgml myoglobin was coated ontoan Immunlon-2 96-well plate and incubated overnight The plate was then washed and TMB added for 1 hour to promote oxidation Theoxidizedmaterial was then added to a fresh well containing either water or 2mMPIC1 Samples were thenmeasured by absorbance at 450 nmData are means of three independent experiments plusmn SEM

pathological conditions such as sickle cell disease ischemia-reperfusion injury malaria hemolytic anemia blood trans-fusion and renal failure [5 6] Rhabdomyolysis results in therelease of toxic myoglobin [19] While haptoglobin can bindsome free Hb and heme oxygenase (HO) serves as a hemedetoxification system in the situation of massive free hemeaccumulation these systems are overwhelmed thus allowingfree heme to exert its toxic effects [5] Free heme results in

the formation of ROS that lead to oxidative stress resultingin lipid peroxidation and DNA damage as well as proteinaggregation and damage [2] In addition free heme possessesproinflammatory properties that lead to neutrophil activa-tion which in turn induces chemotaxis cytokine productionand additional ROS as well as HOCl generation [5] Finallyfree heme can bind RBCs and through a number of differentmechanisms cause additional hemolysis [5] Given the highly


Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl

No PIC125 ＧM PIC1125 ＧM PIC10625ＧM PIC1

03125ＧM PIC10156ＧM PIC1

(a)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(b)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(c)

Free

Fe (

uM)

0

2

4

6

8

10

12

RBC lysate

Myoglobin

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

metHb

(d)

Figure 5 PIC1 prevents Hb andmyoglobin destruction and iron release mediated by NaOCl (a) RBC lysates (125 times 107 cellsml equivalents)(b) 02mgmlmetHb and (c) 02mgmlmyoglobin were added to increasing concentrations of PIC1 followed by a 1 1000 dilution of NaOClSamples were then incubated for 5 minutes and spectral absorbance readings were recorded from 300 to 550 nm (d) Free iron (Fe) releasefrom each sample was determined by the ferrozine assay Data are means of three independent experiments plusmn SEM

lethal properties of free heme development of an inhibitorto detoxify the oxidative properties of free heme in acutehemolysis and rhabdomyolysis remainsmajor unmetmedicalneeds

The experiments shown here demonstrate that PIC1 candose-dependently inhibit the peroxidase activity of hemefrom RBC lysates as well as pure metHb and myoglobin forthree different substrates TMB ABTS and O-dianisidineThe level of inhibition was comparable to or better than thatof the MPO inhibitor ABAH Furthermore PIC1 was ableto reverse the oxidation of TMB for RBC lysates metHband myoglobin PIC1 was found to dose-dependently inhibitNaOCl-mediated destruction of heme and prevent the release

of free iron as shown by preservation of the Soret peakin the spectral assay and the ferrozine assay respectivelyInterestingly while ABAH was shown to inhibit Soret peakflattening the inhibitionwas not dose-dependent and did notprevent free iron release suggesting a different mechanismof Hb protection from NaOCl by PIC1 versus ABAH Theinhibition of Hb and myoglobin peroxidase activity areconsistent with our recently published results demonstratingthat PIC1 inhibits peroxidase activity of MPO [9] Togetherthese results suggest that PIC1 can broadly inhibit diverseheme-bearing enzymes

PIC1 derivative PA-dPEG24 contains two vicinal cysteineresidues at positions 9 and 10 It is possible that the observed


Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl

No ABAH25 ＧM ABAH125 ＧM ABAH0625ＧM ABAH

03125ＧM ABAH0156ＧM ABAH

(a)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(b)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(c)

Free

Fe (

uM)

0

2

4

6

8

10

12

14

metHbRBC lysate

Myoglobin

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

(d)

Figure 6 ABAH prevents Hb and myoglobin destruction but does not prevent iron release mediated by NaOCl (a) RBC lysates (125 times 107cellsml equivalents) (b) 02mgml metHb and (c) 02mgml myoglobin were added to increasing concentrations of ABAH followed by a1 1000 dilution of NaOCl Samples were then incubated for 5 minutes and spectral absorbance readings were recorded from 300 to 550 nm(d) Free iron (Fe) release from each sample was determined by the ferrozine assay Data are means of three independent experiments plusmn SEM

inhibition of peroxidase activity for metHb and myoglobinis mediated by the reduction of higher oxidation statesof these proteins by these cysteine residues Oxidation ofthe cysteine residues would be predicted to either resultin intermolecular disulphide bonds between the cysteineresidues of two PIC1 molecules or possibly intramolecularcystine formation PIC1 was found to dose-dependentlyinhibit peroxidase activity of RBC lysates in a similar mannerto L-cysteine (Figure 4(a)) suggesting that these residuesmay mediate the peroxidase inhibitory activity of PIC1 Thepossibility that the cysteine residues of PIC1 facilitate per-oxidase inhibition of these heme-bearing enzymes also may

explain the observed differences in the level of peroxidaseinhibition of ABAH versus PIC1 (Figure 2) as well as Hb andmyoglobin destruction and iron release mediated by NaOCl(Figures 5 and 6) Thiol groups have been documented topossess different reactivity to free radicals and heme highvalent complexes compared to hydrazides like ABAH [20 21]While the cysteine residues of PIC1 are essential for theprevention of peroxidase activity they also are critical for thecomplement inhibitory activity of this peptide as substitutionof either of the two cysteine residues in earlier derivativesof PIC1 inhibited complement activity [22] Experiments arecurrently underway to determine if these cysteine amino


acids exist as free thiols in solution and if the mechanism bywhich PIC1 inhibits peroxidase activity involves oxidation ofone or both of these residues

Free heme is exquisitely toxic to the kidney where itcan lead to renal failure through a number of mechanismsincluding oxidative damage and heme deposition in therenal tubules [5] We have recently developed a rat modelof intravascular hemolytic transfusion reaction in whichWistar rats are transfused with incompatible RBCs [10]The transfused cells lyse releasing free Hb which resultsin acute kidney disease at 6 hours after transfusion asassessed by increased kidney weight and tissue damage byhistopathology Animals prophylactically treated with PIC1showed protection of the kidneys compared to animalsreceiving either saline only or intravenous immunoglobulin(IVIG) [23]We speculate that PIC1may bemediating kidneyprotection in this animal model through two independentanti-inflammatory actions (1) inhibition of complement-mediated RBC lysis preventing free Hb release and (2) inhi-bition of the peroxidase activity of Hb Future experimentsto explore these dual functions of the PIC1 molecule inthis animal model are planned As there are currently nomarketed pharmacological inhibitors for the prevention ofextracellular heme-mediated peroxidation PIC1 may haveutility as a therapeutic agent for diseases involving intravas-cular hemolysis and rhabdomyolysis

5 Conclusions

Free Hb from lysed RBCs and myoglobin released fromdamaged muscle cells are inherently toxic molecules in theblood stream due to their intrinsic peroxidase activity andhave been demonstrated to contribute to pathogenesis inhemolytic diseases and rhabdomyolysis respectively Cur-rently there are no inhibitors of peroxidase activity in themarketplace to address these unmet medical needs PeptideInhibitor of Complement C1 (PIC1) significantly inhibitsperoxidase activity of RBC lysates and purified Hb as wellas myoglobin Based on these findings future experimentsto test PIC1-mediated inhibition of peroxidase activity inpreclinical animal models are currently planned

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This study was supported by Childrenrsquos Health FoundationChildrenrsquos Hospital of The Kingrsquos Daughters Norfolk Vir-ginia USA to Kenji M Cunnion (Grant no 7364)

References

[1] J M Berg J L Tymoczko and L Stryer Eds BiochemistryWH Freeman and Company New York NY USA 2007 WHFreeman and Company

[2] A I Alayash ldquoOxygen therapeutics can we tame haemoglo-binrdquo Nature Reviews Drug Discovery vol 3 no 2 pp 152ndash1592004

[3] D Maitra J Byun P R Andreana et al ldquoMechanism ofhypochlorous acid-mediated heme destruction and free ironreleaserdquo Free Radical Biology and Medicine vol 51 no 2 pp364ndash373 2011

[4] D Maitra J Byun P R Andreana et al ldquoReaction ofhemoglobin with HOCl mechanism of heme destruction andfree iron releaserdquo Free Radical Biology and Medicine vol 51 no2 pp 374ndash386 2011

[5] S Kumar and U Bandyopadhyay ldquoFree heme toxicity and itsdetoxification systems in humanrdquoToxicology Letters vol 157 no3 pp 175ndash188 2005

[6] D J Schaer and P W Buehler ldquoCell-free hemoglobin and itsscavenger proteins new disease models leading the way totargeted therapiesrdquoCold Spring Harbor Perspectives inMedicinevol 3 no 6 2013

[7] J A Sharp P S Hair H K Pallera et al ldquoPeptide inhibitor ofcomplement C1 (PIC1) rapidly inhibits complement activationafter intravascular injection in ratsrdquo PLoS ONE vol 10 no 7Article ID e0132446 2015

[8] J A Sharp P H Whitley K M Cunnion and N K KrishnaldquoPeptide inhibitor of complement C1 a novel suppressor ofclassical pathway activation mechanistic studies and clinicalpotentialrdquo Frontiers in Immunology vol 5 article 406 2014

[9] P S Hair L A Sass N K Krishna and K M CunnionldquoInhibition ofmyeloperoxidase activity in cystic fibrosis sputumby peptide inhibitor of complement C1 (PIC1)rdquo PLoS ONE vol12 no 1 Article ID e0170203 2017

[10] T A Shah C T Mauriello P S Hair et al ldquoComplementinhibition significantly decreases red blood cell lysis in a ratmodel of acute intravascular hemolysisrdquo Transfusion vol 54no 11 pp 2892ndash2900 2014

[11] A Hasmann E Wehrschuetz-Sigl A Marold et al ldquoAnalysisof myeloperoxidase activity in wound fluids as a marker ofinfectionrdquo Annals of Clinical Biochemistry vol 50 no 3 pp245ndash254 2013

[12] N Kothari R S Keshari J Bogra et al ldquoIncreasedmyeloperox-idase enzyme activity in plasma is an indicator of inflammationand onset of sepsisrdquo Journal of Critical Care vol 26 no 4 pp435e1ndash435e7 2011

[13] C T Mauriello H K Pallera J A Sharp et al ldquoA novelpeptide inhibitor of classical and lectin complement activationincludingABO incompatibilityrdquoMolecular Immunology vol 53no 1-2 pp 132ndash139 2013

[14] D Maitra F Shaeib I Abdulhamid et al ldquoMyeloperoxidaseacts as a source of free iron during steady-state catalysisby a feedback inhibitory pathwayrdquo Free Radical Biology andMedicine vol 63 pp 90ndash98 2013

[15] J Huang F Smith and P Panizzi ldquoOrdered cleavage ofmyeloperoxidase ester bonds releases active site heme leadingto inactivation of myeloperoxidase by benzoic acid hydrazideanalogsrdquo Archives of Biochemistry and Biophysics vol 548 pp74ndash85 2014

[16] J Huang F Smith J R Panizzi D C Goodwin and P PanizzildquoInactivation of myeloperoxidase by benzoic acid hydraziderdquoArchives of Biochemistry andBiophysics vol 570 pp 14ndash22 2015

[17] K Juarez-Moreno M Ayala and R Vazquez-Duhalt ldquoAntiox-idant capacity of poly(ethylene glycol) (PEG) as protection


mechanism against hydrogen peroxide inactivation of peroxi-dasesrdquo Applied Biochemistry and Biotechnology vol 177 no 6pp 1364ndash1373 2015

[18] G Atmaca ldquoAntioxidant effects of sulfur-containing aminoacidsrdquo Yonsei Medical Journal vol 45 no 5 pp 776ndash788 2004

[19] L O Chavez M Leon S Einav and J Varon ldquoBeyond muscledestruction a systematic review of rhabdomyolysis for clinicalpracticerdquo Critical Care vol 20 no 1 article 135 2016

[20] A J Kettle C A Gedye and C C Winterbourn ldquoMechanismof inactivation of myeloperoxidase by 4-aminobenzoic acidhydraziderdquo Biochemical Journal vol 321 no 2 pp 503ndash5081997

[21] L B Poole ldquoThe basics of thiols and cysteines in redox biologyand chemistryrdquo Free Radical Biology amp Medicine vol 80 pp148ndash157 2015

[22] J Q Gronemus P S Hair K B Crawford J O NyalwidheK M Cunnion and N K Krishna ldquoPotent inhibition ofthe classical pathway of complement by a novel C1q-bindingpeptide derived from the human astrovirus coat proteinrdquoMolecular Immunology vol 48 no 1-3 pp 305ndash313 2010

[23] P S Kumar H K Pallera P S Hair et al ldquoPeptide inhibitorof complement C1 modulates acute intravascular hemolysis ofmismatched red blood cells in ratsrdquo Transfusion vol 56 no 8pp 2133ndash2145 2016

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


the generation of additional ROS resulting in cellular damageFurther reaction of free iron with H

2O2forms a potent

oxidant that is damaging to lipids nucleic acids and aminoacids Finally Hb protein radical formation can result incrosslinking of heme and amino acids [2] Currently there areno pharmacological agents available on the market to controlpathological extracellular Hb-mediated oxidative reactions

Our laboratory has characterized a family of peptidesknow as Peptide Inhibitor of Complement C1 (PIC1) thatinhibit the classical pathway of complement by bindingthe initiator molecule C1q and inhibiting activation of thecognate serine protease tetramer C1s-C1r-C1r-C1s that isnormally associatedwith C1q [7 8]We have recently demon-strated that the lead pegylated PIC1 compound PA-dPEG24(amino acid sequence IALILEPICCQERAA containing a 24units PEGmoiety attached to the C terminus total molecularweight 2771Da) can inhibit MPO activity in cystic fibrosissputum as well as from neutrophils and purified MPO [9]To ascertain if PIC1 had any inhibitory activity on Hb whichshares with MPO a heme-based mechanism of action weanalyzed the effect of PIC1 on the peroxidase activity of Hbfrom RBCs PIC1 was able to inhibit this activity by Hb aswell as purified metHb and myoglobin and also protect itsdestruction by sodiumhypochlorite (NaOCl) suggesting thatPIC1 possesses a novel antioxidant activity that may be oftherapeutic benefit

2 Materials and Methods

21 Ethics Statement Human blood was obtained from fourhealthy volunteers for the generation of RBC lysates usedin these studies per the Eastern Virginia Medical SchoolIRB approved protocol 02-06-EX-0216 The study partic-ipants provided written informed consent Human bloodwas collected for these experiments from May 2016 throughNovember 2016

22 Materials PIC1 (IALILEPICCQERAA-dPEG24) wasmanufactured by PolyPeptide Group (San Diego CA) toge95 purity as verified by HPLC and mass spectrometryanalysis Lyophilized PIC1 was solubilized in normal salinewith 001M Na

2HPO4buffer to 15mM dPEG24 was pur-

chased from PolyPeptide Group and dissolved in the samebuffer Pure human methemoglobin (metHb) myoglobinfrom equine skeletal muscle and L-cysteine were purchasedfrom Sigma (St Louis MO) 331015840551015840-Tetramethylbenzidine(TMB) as a solution containing H

2O2was purchased from

Thermo Fisher Scientific (Waltham MA) 4-Aminobenzoicacid hydrazide (ABAH) O-dianisidine and NaOCl werepurchased from Sigma-Aldrich (St Louis MO) 221015840-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) solu-tion was purchased fromThermo Fisher Scientific (WalthamMA)

23 Preparation of Human RBC Lysate RBC lysates wereprepared from the blood of healthy donors as previouslydescribed [10] Cells were washed twice with GVBSminusminus(veronal-buffered saline 01 gelatin and 001M EDTA) andthen incubated at 37∘C to allow for spontaneous lysis The

cells were then washed twice again with GVBS++ (veronal-buffered saline 015mM CaCl

2 and 10mM MgCl

2) and

resuspended in a volume which set the concentration to 1times 109 cellsml (absorbance reading of lysed RBCs at 541 nmof 0210 = 1 times 109 cellsml) An aliquot of these cells wascombinedwith an equal aliquot of water at room temperatureand spun at 21000timesg for 5 minutes to separate the releasedhemoglobin from the cell debris

24 Measurement of Peroxidase Inhibition by PIC1 Inhibi-tion of peroxidase activity was assessed by combining theRBC lysate (reported in cellular equivalentsml) metHb(05mgml) or myoglobin (05mgml) with increasing con-centrations of PIC1 with a constant final volume for 5minutes before adding TMB as described below Additionallyincreasing amounts of RBC lysates were incubated withincreasing amounts of PIC1 with a constant final volume for5 minutes before adding TMB As a positive control for inhi-bition of peroxidase activity increasing amounts of ABAHor L-cysteine were included in select experiments After the5-minute incubation samples were combined with 100120583l ofTMB in a nontreated 96-well plate for 15 seconds followedby 100 120583l of 25N H

2SO4(stop buffer) and absorbance was

read at 450 nm in a Synergy HT BioTek plate reader [10] Forassays usingABTS 150 120583l of ABTS solutionwas added to eachsample and incubated for 15 seconds before adding 100120583lof 1 SDS to stop the reaction [11] Absorbance was readat 405 nm O-Dianisidine working solution was prepared bymaking a 0166mgml solution in 50mM phosphate-citratebuffer pH 50with a final of 0006H

2O2added immediately

before use [12] 100 120583l of the prepared O-dianisidine solutionwas added to each sample and incubated for 2 minutes beforeadding 100 120583l of 25NH

2SO4to stop the reactionAbsorbance

was read at 405 nmTo assess if PIC1 could inhibit peroxidase activity of Hb

released from hemolyzed RBCs in human serum type Osera at 15 in GVBS++ buffer was mixed with type ABRBCs at titrating concentrations (1 2 dilutions of 5 times 107RBCs) followed by incubation for 1 hour at 37∘C to allow forRBC hemolysis via antibody mediated complement lysis [13]Samples were then centrifuged and supernatant containingthe released Hb collected PIC1 at 1mM final concentrationwas subsequently combined with the sample supernatantsand allowed to incubate for 5 minutes before adding TMBand H

2SO4as above to evaluate peroxidase activity from the

cell-free Hb

25 Reversal of Peroxidase Activity by PIC1 RBC lysate(25 times 108 cellsml equivalent) 2mgml metHb or 2mgmlmyoglobin was coated onto an Immunlon-2 96-well plate ina carbonate coating buffer (016M Na

2CO3 034 NaHCO

3

pH 96) overnight at 4∘C The plate was then washed TMBwas added and allowed to incubate for 1 hour The oxidizedTMBwas transferred to a freshwell containing eitherwater or2mM PIC1 The reaction was stopped after 1min with 25MH2SO4 Absorbance was read at 450 nm

26 Free IronAssay Iron release from theRBC lysatemetHbor myoglobin was measured utilizing a ferrozine assay [14]





3 Results









PIC1 (mM)0 1 2 3 4 5 6 7 8

00

05

10

15

20

25

30

35

40

Donor 1Donor 2

Donor 3Donor 4

Abs (

450Ｈ

m)

(a)

PIC1 (mM)00 05 10 15 20 25 30 35 40

00

05

10

15

20

25

30

35

40






Abs (

450Ｈ

m)

(b)

0 1 2 3 4 500

05

10

15

20

25

30

35

No PIC11ＧM PIC1

RBC (times107)

Abs (

412Ｈ

m)

(c)







00

04

08

12

16

20

PIC1ABAH

Abs (

450Ｈ

m)

(a)


00

05

10

15

20

25

30

35

PIC1ABAH

Abs (

450Ｈ

m)

(b)


00

02

04

06

08

10

PIC1ABAH

Abs (

450Ｈ

m)

(c)


00

05

10

15

20

PIC1PEG24

Abs (

450Ｈ

m)

(d)





4 Discussion



PIC1ABAH


00

05

10

15

20

25

30Ab

s (405Ｈ

m)

(a)

PIC1ABAH


00

02

04

06

08

10

12

14

Abs (

405Ｈ

m)

(b)


Inhibitor (mM)0 1 2 3 4 5 6 7 8

00

05

10

15

20

25

30

35

40

CysteinePIC1

Abs (

450Ｈ

m)

(a)

00

01

02

03

04

05


Wat

er

Wat

er

Wat

er

PIC1

PIC1

PIC1

Abso

rban

ce (4

50Ｈ

m)

(b)





Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(a)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(b)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(c)

Free

Fe (

uM)

0

2

4

6

8

10

12

RBC lysate

Myoglobin

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

metHb

(d)







Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(a)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(b)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(c)

Free

Fe (

uM)

0

2

4

6

8

10

12

14

metHbRBC lysate

Myoglobin

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

(d)







5 Conclusions




Acknowledgments


References

































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





3 Results









PIC1 (mM)0 1 2 3 4 5 6 7 8

00

05

10

15

20

25

30

35

40

Donor 1Donor 2

Donor 3Donor 4

Abs (

450Ｈ

m)

(a)

PIC1 (mM)00 05 10 15 20 25 30 35 40

00

05

10

15

20

25

30

35

40






Abs (

450Ｈ

m)

(b)

0 1 2 3 4 500

05

10

15

20

25

30

35

No PIC11ＧM PIC1

RBC (times107)

Abs (

412Ｈ

m)

(c)







00

04

08

12

16

20

PIC1ABAH

Abs (

450Ｈ

m)

(a)


00

05

10

15

20

25

30

35

PIC1ABAH

Abs (

450Ｈ

m)

(b)


00

02

04

06

08

10

PIC1ABAH

Abs (

450Ｈ

m)

(c)


00

05

10

15

20

PIC1PEG24

Abs (

450Ｈ

m)

(d)





4 Discussion



PIC1ABAH


00

05

10

15

20

25

30Ab

s (405Ｈ

m)

(a)

PIC1ABAH


00

02

04

06

08

10

12

14

Abs (

405Ｈ

m)

(b)


Inhibitor (mM)0 1 2 3 4 5 6 7 8

00

05

10

15

20

25

30

35

40

CysteinePIC1

Abs (

450Ｈ

m)

(a)

00

01

02

03

04

05


Wat

er

Wat

er

Wat

er

PIC1

PIC1

PIC1

Abso

rban

ce (4

50Ｈ

m)

(b)





Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(a)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(b)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(c)

Free

Fe (

uM)

0

2

4

6

8

10

12

RBC lysate

Myoglobin

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

metHb

(d)







Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(a)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(b)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(c)

Free

Fe (

uM)

0

2

4

6

8

10

12

14

metHbRBC lysate

Myoglobin

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

(d)







5 Conclusions




Acknowledgments


References

































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


PIC1 (mM)0 1 2 3 4 5 6 7 8

00

05

10

15

20

25

30

35

40

Donor 1Donor 2

Donor 3Donor 4

Abs (

450Ｈ

m)

(a)

PIC1 (mM)00 05 10 15 20 25 30 35 40

00

05

10

15

20

25

30

35

40






Abs (

450Ｈ

m)

(b)

0 1 2 3 4 500

05

10

15

20

25

30

35

No PIC11ＧM PIC1

RBC (times107)

Abs (

412Ｈ

m)

(c)







00

04

08

12

16

20

PIC1ABAH

Abs (

450Ｈ

m)

(a)


00

05

10

15

20

25

30

35

PIC1ABAH

Abs (

450Ｈ

m)

(b)


00

02

04

06

08

10

PIC1ABAH

Abs (

450Ｈ

m)

(c)


00

05

10

15

20

PIC1PEG24

Abs (

450Ｈ

m)

(d)





4 Discussion



PIC1ABAH


00

05

10

15

20

25

30Ab

s (405Ｈ

m)

(a)

PIC1ABAH


00

02

04

06

08

10

12

14

Abs (

405Ｈ

m)

(b)


Inhibitor (mM)0 1 2 3 4 5 6 7 8

00

05

10

15

20

25

30

35

40

CysteinePIC1

Abs (

450Ｈ

m)

(a)

00

01

02

03

04

05


Wat

er

Wat

er

Wat

er

PIC1

PIC1

PIC1

Abso

rban

ce (4

50Ｈ

m)

(b)





Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(a)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(b)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(c)

Free

Fe (

uM)

0

2

4

6

8

10

12

RBC lysate

Myoglobin

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

metHb

(d)







Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(a)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(b)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(c)

Free

Fe (

uM)

0

2

4

6

8

10

12

14

metHbRBC lysate

Myoglobin

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

(d)







5 Conclusions




Acknowledgments


References

































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



00

04

08

12

16

20

PIC1ABAH

Abs (

450Ｈ

m)

(a)


00

05

10

15

20

25

30

35

PIC1ABAH

Abs (

450Ｈ

m)

(b)


00

02

04

06

08

10

PIC1ABAH

Abs (

450Ｈ

m)

(c)


00

05

10

15

20

PIC1PEG24

Abs (

450Ｈ

m)

(d)





4 Discussion



PIC1ABAH


00

05

10

15

20

25

30Ab

s (405Ｈ

m)

(a)

PIC1ABAH


00

02

04

06

08

10

12

14

Abs (

405Ｈ

m)

(b)


Inhibitor (mM)0 1 2 3 4 5 6 7 8

00

05

10

15

20

25

30

35

40

CysteinePIC1

Abs (

450Ｈ

m)

(a)

00

01

02

03

04

05


Wat

er

Wat

er

Wat

er

PIC1

PIC1

PIC1

Abso

rban

ce (4

50Ｈ

m)

(b)





Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(a)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(b)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(c)

Free

Fe (

uM)

0

2

4

6

8

10

12

RBC lysate

Myoglobin

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

metHb

(d)







Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(a)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(b)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(c)

Free

Fe (

uM)

0

2

4

6

8

10

12

14

metHbRBC lysate

Myoglobin

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

(d)







5 Conclusions




Acknowledgments


References

































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


PIC1ABAH


00

05

10

15

20

25

30Ab

s (405Ｈ

m)

(a)

PIC1ABAH


00

02

04

06

08

10

12

14

Abs (

405Ｈ

m)

(b)


Inhibitor (mM)0 1 2 3 4 5 6 7 8

00

05

10

15

20

25

30

35

40

CysteinePIC1

Abs (

450Ｈ

m)

(a)

00

01

02

03

04

05


Wat

er

Wat

er

Wat

er

PIC1

PIC1

PIC1

Abso

rban

ce (4

50Ｈ

m)

(b)





Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(a)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(b)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(c)

Free

Fe (

uM)

0

2

4

6

8

10

12

RBC lysate

Myoglobin

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

metHb

(d)







Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(a)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(b)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(c)

Free

Fe (

uM)

0

2

4

6

8

10

12

14

metHbRBC lysate

Myoglobin

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

(d)







5 Conclusions




Acknowledgments


References

































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(a)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(b)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(c)

Free

Fe (

uM)

0

2

4

6

8

10

12

RBC lysate

Myoglobin

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

metHb

(d)







Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(a)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(b)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(c)

Free

Fe (

uM)

0

2

4

6

8

10

12

14

metHbRBC lysate

Myoglobin

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

(d)







5 Conclusions




Acknowledgments


References

































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(a)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(b)

Wavelength (nm)300 350 400 450 500 550 600

Abso

rban

ce

00

01

02

03

04

05

No NaOCl



(c)

Free

Fe (

uM)

0

2

4

6

8

10

12

14

metHbRBC lysate

Myoglobin

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

No

NaO

Cl

0Ｇ

M0156Ｇ

M03125Ｇ

M0625Ｇ

M125Ｇ

M25

ＧM

(d)







5 Conclusions




Acknowledgments


References

































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




5 Conclusions




Acknowledgments


References

































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

peptide inhibitor of complement c1 inhibits the peroxidase...

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