quantitativeassessment of the effects of reducing agents...

Research ArticleQuantitative Assessment of the Effects of Reducing Agentson Biological Macromolecules and on the Possible Repair ofOxidative Damage

Jianan Zhao1 Naijin Xu2 and Hui Liu 1

1College of Medical Laboratory Dalian Medical University Dalian 116044 China2Department of Chemistry Dalian Medical University Dalian 116044 China

Correspondence should be addressed to Hui Liu immunologydlmedueducn

Received 27 March 2018 Accepted 19 July 2018 Published 2 August 2018

Academic Editor Xudong Huang

Copyright copy 2018 Jianan Zhao 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

Objective To quantitatively assess the influence of reducing agents on biological macromolecules and on the possible repair ofoxidative damageMethods Samples (antibody enzyme DNA and diluted serum) were treated with reducing agents (ammoniumferrous sulfate ascorbic acid potassium iodide and sodium hyposulfite) in the experimental group and with NaCl in the controlgroup Enzyme-linked immunosorbent assay and quantitative PCR were used to determine the activity of antibody enzyme andDNA Native gel electrophoresis (Native-PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) wereused to determine protein structure Reducing agents that had no inhibitory effect on biological macromolecules were selectedAntibodies were treated with oxidants to caused oxidative damage and then treated with reducing agents and the possible repairof oxidative damage was assessed Results Certain concentrations of ammonium ferrous sulfate resulted in significant inhibition ofantibody enzyme DNA and diluted serum Certain concentrations of ascorbic acid resulted in significant inhibition of antibodySodium hyposulfite and potassium iodide had no effect on antibody enzyme DNA and diluted serumThe OD values in group A(in whichHBsAb was treated by oxidation and then a reductant) were significantly higher than those in group B (HBsAb treated byoxidation) Conclusion Ammonium ferrous sulfate ascorbic acid sodium hyposulfite and potassium iodide had different effectson antibody enzyme DNA and diluted serum The reduction in antibody activity due to an oxidant was partially repaired by areductant

1 Introduction

Oxidative stress is an imbalance between oxidation andantioxidation in the body which tends to oxidation [1ndash3] Negative effects are produced in the body by oxidizingsubstances and this is thought to be an important factor inaging and disease [2 4 5]The structures of various biologicalmacromolecules such as proteins lipids carbohydrates andDNA are altered by oxidation [3 5ndash7] Modification ofamino acid residues is the main cause of protein damage andmay result in breakage hydrolysis peptide crosslinking andpolymerization which lead to changes in the structure andconformation thereby affecting the biological activity of theprotein [1 6]

Antioxidation is the process of slowing down or pre-venting oxidation [3 8 9] Substances that interfere with

the initiation and diffusion of free radical chain reactionsor inhibit the reactions of free radicals are called antioxi-dants Antioxidants are usually reductants Antioxidants arethought to counteract the effects of oxidants and reduceoxidative damage to the body [3 5] However research onthe possible repair of oxidative damage by reducing agentshas rarely been reported This study attempts to investigatethe possible repair of oxidative damage in proteins from theviewpoint of chemistry

Many researchers believe that high concentrations ofantioxidants in tissues are good for the body but someresearchers are skeptical [5 10 11] Some studies have foundthat there are some chemical side effects of reducing agents[10 12 13] Therefore to investigate the repair of oxida-tive damage reducing agents with no inhibitory effects onbioactive molecules should be selected first In this study

HindawiBioMed Research InternationalVolume 2018 Article ID 5704016 7 pageshttpsdoiorg10115520185704016

2 BioMed Research International

samples (antibody enzyme DNA and diluted serum) weretreated with well-known reducing agents (ammonium fer-rous sulfate ascorbic acid sodiumhyposulfite and potassiumiodide) in the experimental group and with NaCl in thecontrol group The reducing agents (sodium hyposulfite andpotassium iodide) that had no inhibitory effect on biologicalmacromolecules were selected with using enzyme-linkedimmunosorbent assay (ELISA) and quantitative PCR (qPCR)to determine the activity of antibody enzyme and DNAAntibodies were treated with oxidants to cause oxidativedamage and then treated with reducing agents and thepossible repair of oxidative damage was assessed We believethat biological activity can be recovered following oxidativedamage using safe reducing agents which is the experimentalfoundation for antioxidant treatments in vitro andor in vivo

2 Materials and Methods

21 Effects of Reducing Agents on Antibody Activity

211 Reducing agents (6mmolL ammonium ferrous sulfate6mmolL ascorbic acid 60mmolL sodium hyposulfite and60mmolL potassium iodide) were diluted at a 11 ratio usingdistilled water to obtain eight concentrations NaCl solutionswere prepared with the same pH and ionic strength as thereductant solvent

212The samples (HBsAb healthy humanmixed serumwithan antibody to hepatitis B surface antigen collected froma hospital in Dalian China) were mixed with a reductantsolution or NaCl solution in a test tube at a ratio of 15 at 4∘Cfor 1 h

213 The antibody activity was detected with ELISA (hep-atitis B virus surface antibody test kit Chen Yu China) TheOD values were used to assess antibody activity with higherOD values indicating higher antibody activity The OD valuefor the blank was 0042 which is eliminated in the followingcalculation Inhibition rate = [(the OD value of the controlgroup ndash theODvalue of the blank hole) ndash (theODvalue of theexperimental group ndash the OD value of the blank hole)](theOD value of the control group ndash the OD value of the blankhole)times100 Data were analyzed using GraphPad Prism 5and are presented as the mean plusmn SD Statistical significancewas determined using Studentrsquos 119905-test and is presented aslowast(119901 lt 005) lowastlowast(119901 lt 001) or lowastlowastlowast(119901 lt 0001)

22 Effects of the Reducing Agents on Protein Structures

221Reducing agents were prepared at the following concen-trations 78mmolL ammonium ferrous sulfate 78mmolLascorbic acid 78mmolL sodium hyposulfite and 78mmolLpotassium iodide and NaCl solutions were prepared at theconcentrations of 78 and 78mmolL

222 The diluted serum (healthy human serum collectedfrom a hospital in Dalian China) was mixed with a reductantsolution or NaCl solution in a test tube at a ratio of 14 at 4∘Cfor 1 h and protected from light

223 Native-PAGE and SDS-PAGE were used to investigatewhether the spatial structures and molecular structuresof the proteins had changed [14ndash16] Separation gels andconcentrated gels were established at 12 and 5 Concen-trated gels were electrophoresed for 35min separation gelswere electrophoresed for 80min followed by staining withCoomassie Brilliant Blue and then decolorized

23 Effects of the Reducing Agents on the Activity ofTaq Enzyme and DNA

231 Reducing agents (10mmolL ammonium ferrous sul-fate 10mmolL ascorbic acid 100mmolL sodium hyposul-fite and 100mmolL potassium iodide) were diluted at a11 ratio using distilled water to obtain eight concentrationsNaCl solutions were prepared with the same pH and ionicstrength as the reductant solvent The Taq enzyme (SYBRPremix Ex Taq TMII Tli RNaseH Plus Takara Japan) wasmixed with a reductant solution or NaCl solution in a testtube at a ratio of 11 and the solutions were placed for 1 hand protected from light Other ingredients were not treatedexcept enzymes

232Reducing agents (6mmolL ammonium ferrous sulfate6mmolL ascorbic acid 60mmolL sodium hyposulfite and60mmolL potassium iodide) were diluted at a 11 ratiousing distilled water to obtain eight concentrations NaClsolutions were prepared with the same pH and ionic strengthas the reductant solvent DNA samples were extracted usinga DNA extraction kit (Omega Biotek Norcross GA USA)The primers for genes used in this study were szlig-actin (F51015840-CAAATATGAGATGCGTTGTTCAGG-31015840 R51015840-TGTGTG-GACTTGGGAGAGGA-31015840 ) and CRP (F51015840-AATGTGAAC-ATGTGGGACTTTGTG-31015840 R51015840-CGCCAGTTCAGGACA-TTAGGAC-31015840) The DNA was mixed with a reductantsolution orNaCl solution in a test tube at a ratio of 15 and thesolutions were placed for 1 h and protected from light Otheringredients were not treated except DNA

233 Ct values were determined using qPCR and corre-sponding Ct values for the same gene in the experimentalgroup and the control group were recorded Inhibition rateswere calculated using the following equation Inhibition rate= (Ct value of the experimental group ndash Ct value of thecontrol group)Ct value of the control group times100 Datawere analyzed using GraphPad Prism 5 and are presented asthe mean plusmn SD Statistical significance was determined usingStudentrsquos 119905-test and is presented as lowast(119901 lt 005) lowastlowast(119901 lt 001)or lowastlowastlowast(119901 lt 0001)

24 Possible Reversion of Oxidative Damage

241 Reductants with no significant inhibitory effects onthe activity of the antibody enzyme DNAand proteinstructures were selected via the procedures in Sections 2122 and 23 The antibody to HBsAg was treated withpotassium hypermanganate (25mmolL) to cause oxidativedamage [17] After removing the oxidant the antibody wastreated with reductant (50mmolL sodium hyposulfite and

BioMed Research International 3

Group A

Group B

Group C

Group D

Wash HBsAb was treatedwith reductant

HBsAb was treatedwith reductant

HBsAb was treatedwith NaCl



HBsAb was treated

HBsAb was treated

HBsAb was treated

with NaCl

Wash

Wash

Wash

Wash

Washe ODvalues weredetected by

Processingof data

ELISAWash

Wash

with ＋2－Ｈ4

with ＋2－Ｈ4

Figure 1 The grouping method and sample processing methods

50mmolL potassium iodide the experimental concentra-tion was obtained from a previous experiment) The exper-iment included four groups group A group B group Cand group D The grouping method and sample processingmethods in each group are shown in Figure 1

242 Hepatitis B surface antigen samples (recombinanthepatitis B vaccine Han Xin Biopharmaceutical CompanyDalian China) (1100 dilution) were added to the plate andtheOD values were determined with ELISA (hepatitis B virussurface antigen test kit Chen Yu China) The OD valuesin group A group B group C and group D were recordedData were analyzed using GraphPad Prism 5 Statisticalsignificance was determined using Studentrsquos 119905-test and ispresented as the mean plusmn SD and 119875 value

3 Results

31The Effects of Reductants on Antibody The inhibitory ratein the experimental group was significantly different to thatin the control group when the concentration of ammoniumferrous sulfate was 5 25 125 0625 and 03125mmolLThe inhibitory rate in the experimental group was notsignificantly different to that in the control group when theconcentration of ammonium ferrous sulfate was 0156250078125 and 00390625mmolL (Table 1) The inhibitoryrate in the experimental group was significantly different tothat in the control group when the ascorbic acid concentra-tion was 5 25 125 and 0625mmolL The inhibitory ratein the experimental group was not significantly different tothat in the control group when the ascorbic acid concen-tration was 03125 015625 0078125 and 00390625mmolL(Table 1) The inhibitory rate in the experimental groupwas not significantly different to that in the control groupwhen the concentration of potassium iodide and sodiumhyposulfite were 50 25 125 625 3125 15625 078125 and0390625mmolL (Table 2)

32 The Effects of Reductants on the Structure of SerumProteins The serum protein bands treated with ammoniumferrous sulfate were deficient and fuzzy compared withthose treated with NaCl The serum proteins treated withascorbic acid potassium iodide and sodium hyposulfite wereunchanged compared with those treated with NaCl (Figures2 and 3)

Table 1 Inhibitory rates of different concentrations of ammoniumferrous sulfate and ascorbic acid on antibodies ()

Concentration of reducingagents (mmolL)

ammonium ferroussulfate ascorbic acid

5 5104plusmn080lowastlowastlowast 3112plusmn500lowastlowastlowast


125 3583plusmn096lowastlowastlowast 1175plusmn297lowastlowast

0625 1059plusmn295lowastlowast 843plusmn104lowast

03125 650plusmn232lowast 302plusmn178015625 267plusmn297 -136plusmn2970078125 -094plusmn210 095plusmn20200390625 138plusmn070 248plusmn154Statistical significance was determined using the Studentrsquos 119905-test and ispresented as lowast(119901 lt 005) lowastlowast(119901 lt 001) or lowastlowastlowast(119901 lt 0001)

Table 2 Inhibitory rates of different concentrations of potassiumiodide and sodium hyposulfite on antibodies ()

Concentration of reducingagents (mmolL) potassium iodide sodium

hyposulfite50 045 plusmn 222 072 plusmn 20825 498 plusmn 426 minus039 plusmn 179125 304 plusmn 206 minus136 plusmn 332625 125 plusmn 040 090 plusmn 3153125 410 plusmn 193 074 plusmn 03115625 328 plusmn 204 157 plusmn 083078125 minus202 plusmn 082 049 plusmn 1150390625 187 plusmn 086 minus002 plusmn 126

33 The Effects of Reductants on Taq Enzyme The inhibitoryrate in the experimental group was significantly differentto that in the control group when the concentration ofammonium ferrous sulfate was 5 25 125 and 0625mmolL(Table 3) The inhibitory rate in the experimental groupwas not significantly different to that in the control groupwhen the concentration of ammonium ferrous sulfate was03125 015625 0078125 and 00390625mmolL (Table 3)The inhibitory rate in the experimental group was not signifi-cantly different to that in the control group when the ascorbicacid concentration was 5 25 125 0625 03125 0156250078125 and 00390625mmolL (Table 3) The inhibitoryrate in the experimental group was not significantly different


Table 3 Inhibitory rates of different concentrations of ammonium ferrous sulfate and ascorbic acid on Taq enzyme ()

Concentration of reducing agents(mmolL) ammonium ferrous sulfate ascorbic acidszlig-actin CRP szlig-actin CRP

5 6763plusmn110lowastlowastlowast 7577plusmn080lowastlowastlowast -006plusmn014 -088plusmn22125 6409plusmn173lowastlowastlowast 7259plusmn211lowastlowastlowast 060plusmn136 210plusmn226125 6442plusmn344lowastlowastlowast 7041plusmn039lowastlowastlowast 382plusmn124 -006plusmn4160625 1526plusmn301lowastlowast 1359plusmn265lowast 036plusmn404 -114plusmn2803125 396plusmn058 715plusmn432 241plusmn075 -212plusmn475015625 333plusmn201 543plusmn082 271plusmn675 -080plusmn1840078125 003plusmn250 319plusmn213 -217plusmn421 -245plusmn21600390625 017plusmn152 425plusmn150 -225plusmn485 301plusmn527Statistical significance was determined using the Studentrsquos 119905-test and is presented as lowast(119901 lt 005) lowastlowast(119901 lt 001) or lowastlowastlowast(119901 lt 0001)

Figure 2 The effects of reductants on the spatial structure ofproteinsmdashNative-PAGE

Figure 3 The effects of reductants on protein molecularstructuresmdashSDS-PAGE

to that in the control group when the concentrations ofpotassium iodide and sodium hyposulfite were 50 25 125625 3125 15625 078125 and 0390625mmolL (Table 4)

34 The Effects of Reductants on DNA The inhibitory ratein the experimental group was significantly different to thatin the control group when the concentration of ammoniumferrous sulfate was 5 25 125 0625 03125 015625 0078125

and 00390625mmolL (Table 5) The inhibitory rate inthe experimental group was not significantly different tothat in the control group when the ascorbic acid concen-tration was 5 25 125 0625 03125 015625 0078125and 00390625mmolL (Table 5) The inhibitory rate in theexperimental group was not significantly different to thatin the control group when the concentrations of potassiumiodide and sodium hyposulfite were 50 25 125 625 312515625 078125 and 0390625mmolL (Table 6)

35 The Possible Repair of Oxidative Damage The resultsof Sections 31 32 33 and 34 were comprehensively ana-lyzed and the reagents (50mmolL sodium hyposulfite and50mmolL potassium iodide the experimental concentra-tion was obtained from a previous experiment) that had noeffect on the antibody enzyme DNA and protein structureswere selected In this experiment the OD values in group A(HBsAb was treated by oxidation and then a reductant) weresignificantly higher than those in group B (HBsAb treated byoxidation)TheODvalues in group Bwere significantly lowerthan those in group D (HBsAb not treated with a reductantor oxidant) The OD values in group C (HBsAb treated witha reductant) were not significantly different to those in groupDThese results are shown at Tables 7 and 8

4 Discussion

This study comprehensively evaluated the effects of sev-eral reducing agents on the activity of biological macro-molecules On this basis whether the damage to biologicalmacromolecules caused by oxidation can be repaired by areducing agent was investigated Firstly ELISA was usedto analyze the effect of each reductant on antibody activ-ity Hepatitis B surface antibody test kit was used in thisexperiment and the optimal concentrations of reagents anddiluted concentrations of samples were obtained throughpre-experiments Because an antibody is an active proteintemperature pH ionic strength and so on have certaininfluences on its activity [18 19] The ionic strength and pHvalues of the control solutions were in each case matched tothose of the appropriate experimental group by calculationand adjustment and samples of the experimental groupand corresponding control group were added to the sameplate at the same temperature The effects of pH ionic


Table 4 Inhibitory rates of different concentrations of potassium iodide and sodium hyposulfite on Taq enzyme ()

Concentration of reducing agents(mmolL) potassium iodide sodium hyposulfiteszlig-actin CRP szlig-actin CRP

50 397 plusmn 213 214 plusmn 032 724 plusmn 327 288 plusmn 01325 126 plusmn 236 388 plusmn 104 394 plusmn 169 365 plusmn 070125 083 plusmn 118 minus243 plusmn 054 408 plusmn 287 253 plusmn 214625 minus281 plusmn 126 434 plusmn 173 373 plusmn 211 331 plusmn 1353125 223 plusmn 041 153 plusmn 182 398 plusmn 354 003 plusmn 28615625 minus084 plusmn 325 minus092 plusmn 146 354 plusmn 325 131 plusmn 369078125 348 plusmn 157 385 plusmn 004 041 plusmn 135 201 plusmn 1410390625 360 plusmn 235 minus210 plusmn 065 211 plusmn 143 120 plusmn 068

Table 5 Inhibitory rates of different concentrations of ammonium ferrous sulfate and ascorbic acid on DNA ()


5 6681 plusmn 042lowastlowastlowast 7237 plusmn 068lowastlowastlowast minus073 plusmn 046 minus017 plusmn 18125 6121 plusmn 526lowastlowastlowast 7063 plusmn 450lowastlowastlowast 105 plusmn 398 minus214 plusmn 274125 5794 plusmn 199lowastlowastlowast 4081 plusmn 334lowastlowastlowast 045 plusmn 174 042 plusmn 0960625 3952 plusmn 409lowastlowastlowast 3190 plusmn 463lowastlowastlowast minus179 plusmn 111 minus193 plusmn 21503125 2381 plusmn 230lowastlowastlowast 2602 plusmn 185lowastlowastlowast 048 plusmn 033 153 plusmn 226015625 2190 plusmn 050lowastlowast 2382 plusmn 126lowastlowast minus085 plusmn 212 minus048 plusmn 1370078125 1780 plusmn 251lowastlowast 2174 plusmn 466lowastlowast minus145 plusmn 076 213 plusmn 12400390625 1576 plusmn 482lowast 1942 plusmn 341lowastlowast 056 plusmn 043 071 plusmn 152Statistical significance was determined using the Studentrsquos 119905-test and is presented as lowast(119901 lt 005) lowastlowast(119901 lt 001) or lowastlowastlowast(119901 lt 0001)

Table 6 Inhibitory rates of different concentrations of potassium iodide and sodium hyposulfite on DNA ()


50 170 plusmn 043 minus184 plusmn 072 324 plusmn 103 359 plusmn 12925 039 plusmn 067 015 plusmn 169 388 plusmn 042 347 plusmn 074125 069 plusmn 138 057 plusmn 040 248 plusmn 082 188 plusmn 244625 minus158 plusmn 082 196 plusmn 135 410 plusmn 558 113 plusmn 1383125 32 plusmn 155 211 plusmn 189 380 plusmn 024 minus023 plusmn 02815625 286 plusmn 252 minus133 plusmn 054 414 plusmn 469 401 plusmn 383078125 114 plusmn 112 162 plusmn 182 minus175 plusmn 148 minus044 plusmn 1590390625 337 plusmn 244 221 plusmn 244 195 plusmn 150 220 plusmn 031

Table 7 The possible repair of HBsAb oxidative damage bypotassium iodide

Group HBsAb 119901

A 0579 plusmn 0037 00004B 0293 plusmn 0016B 0293 plusmn 0016 00025D 0838 plusmn 0137C 1091 plusmn 0142 00908D 0838 plusmn 0137

strength and temperature on the experimental results werethus eliminated and the experimental results are only relatedto the actions of the reducing agents The results showed that

Table 8The possible repair of HBsAb oxidative damage by sodiumhyposulfite

Group HBsAb 119901


ammonium ferrous sulfate had an inhibitory effect on theactivity of antibodies at concentrations of 5mmolL 25 1250625 and 03125mmolL ascorbic acid had an inhibitory


effect on the activity of antibodies at concentrations of 525 125 and 0625mmolL potassium iodide and sodiumhyposulfite had no inhibitory effects on the activity ofantibodies in the concentration range used in the experiment

Secondly native-PAGE and SDS-PAGE were used tocompare the protein bands of the experimental group andcontrol group on the same gel and to explore whether thespatial structures and molecular structures of the proteinshad changedTherewere no reductive substances in the gel orbuffer for native-PAGE and each sample was directly spottedfor electrophoresis without heating or degeneration so thatthe proteins maintained their natural biological activity andwere separated according to their natural shapes and charges[14 15] The results showed that the protein bands in theammonium ferrous sulfate group were deficient and fuzzycompared with the NaCl group suggesting that the naturalstructures of the proteins were damaged by ammoniumferrous sulfate The protein bands for the ascorbic acidpotassium iodide and sodium hyposulfite groups showed nodifferences compared with the NaCl group indicating thatthe natural structures of the proteins were not destroyed byascorbic acid potassium iodide or sodium hyposulfite InSDS-PAGE the gel and buffer contain a strong reductivematerial (SDS) The proteins of the samples are heated anddenatured and separated according to the molecular weightsof the protein subunits [16 20] The results showed thatthe protein bands in the ammonium ferrous sulfate groupwere deficient and fuzzy compared with the NaCl groupsuggesting that the subunit structures of the proteins weredamaged by ammonium ferrous sulfateThe protein bands ofthe ascorbic acid potassium iodide and sodium hyposulfitegroups were the same as those of the NaCl group indicatingthat ascorbic acid potassium iodide and sodium hyposulfitedid not destroy the subunit structures of the proteins

Thirdly the effects of the reducing agents on Taq enzymeactivity and DNA structure were investigated with qPCRComparing the experimental group to the control groupthe pH value ionic strength and temperature were allconsistent so the experimental results were mainly relatedto the reducing agent In the same reaction system theexperimental group of the same gene was compared with thatof the control group The increase in the Ct value indicatedthat amplification was inhibited suggesting that the reducingagent had an effect on the enzyme or DNA The resultsshowed that ammonium ferrous sulfate had an inhibitoryeffect on the activity of enzymes and DNA in the range of00390625 to 5mmolL ascorbic acid potassium iodide andsodium hyposulfite had no inhibitory effect on the activityof enzymes and DNA in the concentration range used in theexperiment

The foregoing results showed that potassium iodide andsodium hyposulfite had no obvious inhibitory effects on theactivities of the antibody or enzymes or DNA and proteinstructure Potassium iodide and sodium hyposulfite wereselected for the reversion test of oxidative damage Theexperimental concentrationwas themaximumconcentrationthat had no effect on the antibody in a previous experiment(50mmolL sodium hyposulfite and 50mmolL potassiumiodide)

The final experiment was the possible repair of oxidativedamage The oxidative damage model was prepared usingpotassium permanganate The antibody was damaged bythe oxidant and then treated with reductant and the anti-body activity (OD values) was measured with ELISA Thisexperiment was divided into four groups A B C and Dand the OD values of the four groups were compared Thedamage caused by K2MnO4 to the antibody was analyzedby comparing groups B and D [17] The results showed thatthe OD values in group B were significantly lower thanthose in D group indicating that the antibody activity wasobviously damaged by potassium hypermanganate GroupC was compared with group D to investigate the effect ofreducing agent on antibody activity The results showed thatthe OD values in group C were not significantly different tothose in group DThis suggested that the reducing agent hadno significant effect on the activity of antibody Group A wascompared with group B to investigate whether the reducingagent could repair oxidative damage The results showed thatthe ODvalues in groupAwere significantly higher than thosein group B This suggested that the reducing agent had arepairing effect on antibody damage caused by the oxidantIt is found that the reducing agent had only a partial repaireffect on this damage by comparing OD values

We comprehensively evaluated the effects of severalreducing agents on the activity of biological macromoleculesThese findings have some implications for the benefits anddisadvantages of antioxidant supplementation We found thatreduced antibody activity due to an oxidant can be partiallyrepaired by a reductant Further studies on the specificmechanisms involved in oxidative stress and injury repairwould be beneficial

Data Availability

The data used to support the findings of this study areincluded within the article

Conflicts of Interest

The authors declare that they have no conflicts of interest

References

[1] E S Cannizzo C C Clement R Sahu C Follo and L San-tambrogio ldquoOxidative stress inflamm-aging and immunose-nescencerdquo Journal of Proteomics vol 74 no 11 pp 2313ndash23232011

[2] A Agarwal S Gupta andR K Sharma ldquoRole of oxidative stressin female reproductionrdquo Reproductive Biology and Endocrinol-ogy vol 14 no 3 article 28 2005

[3] D Fusco G Colloca M R Lo Monaco andM Cesari ldquoEffectsof antioxidant supplementation on the aging processrdquo ClinicalInterventions in Aging vol 2 no 3 pp 377ndash387 2007

[4] M de La Fuente and J Miquel ldquoAn update of the oxidation-inflammation theory of aging the involvement of the immunesystem inOxi-Inflamm-AgingrdquoCurrent Pharmaceutical Designvol 15 no 26 pp 3003ndash3026 2009


[5] M Bonnefoy J Drai andT Kostka ldquoAntioxidants to slow agingfacts and perspectivesrdquo La Presse Medicale vol 31 no 25 pp1174ndash1184 2002

[6] N Khansari Y Shakiba and M Mahmoudi ldquoChronic inflam-mation and oxidative stress as a major cause of age-relateddiseases and cancerrdquo Recent Patents on Inflammation amp AllergyDrug Discovery vol 3 no 1 pp 73ndash80 2009

[7] PMecocciUMacGarvey andM F Beal ldquoOxidative damage tomitochondrial DNA is increased inAlzheimerrsquos diseaserdquoAnnalsof Neurology vol 36 no 5 pp 747ndash751 1994

[8] J K Willcox S L Ash and G L Catignani ldquoAntioxidants andprevention of chronic diseaserdquo Critical Reviews in Food Scienceand Nutrition vol 44 no 4 pp 275ndash295 2004

[9] M De la Fuente ldquoEffects of antioxidants on immune systemageingrdquo European Journal of Clinical Nutrition vol 56 supple-ment 3 pp S5ndashS8 2002

[10] I Sadowska-Bartosz and G Bartosz ldquoEffect of AntioxidantsSupplementation on Aging and Longevityrdquo BioMed ResearchInternational vol 2014 Article ID 404680 17 pages 2014

[11] V Calabrese D A Butterfield and A M Stella ldquoNutritionalantioxidants and the heme oxygenase pathway of stress toler-ance novel targets for neuroprotection in Alzheimerrsquos diseaserdquoThe Italian journal of biochemistry vol 52 no 4 pp 177ndash1812003

[12] H E Seifried S S McDonald D E Anderson P Greenwaldand J A Milner ldquoThe antioxidant conundrum in cancerrdquoCancer Research vol 63 no 15 pp 4295ndash4298 2003

[13] R I Salganik ldquoThe benefits and hazards of antioxidants con-trolling apoptosis and other protective mechanisms in cancerpatients and the human populationrdquo Journal of the AmericanCollege of Nutrition vol 20 supplement 5 pp 464Sndash472S 2001

[14] V Reisinger and L A Eichacker ldquoAnalysis ofmembrane proteincomplexes by blue native PAGErdquo Proteomics vol 1 no 1-2 pp6ndash15 2006

[15] J Dresler J Klimentova and J Stulik ldquoBacterial protein com-plexes investigation using blue native PAGErdquo MicrobiologicalResearch vol 166 no 1 pp 47ndash62 2011

[16] J Dresler J Klimentova and J Stulik ldquoFrancisella tularensismembrane complexome by blue nativeSDS-PAGErdquo Journal ofProteomics vol 75 no 1 pp 257ndash269 2011

[17] ShuangHan GuanyuWang Naijin Xu andHui Liu ldquoQuantita-tive Assessment of the Effects of Oxidants onAntigen-AntibodyBinding In Vitrordquo Oxidative Medicine and Cellular Longevityvol 2016 Article ID 1480463 7 pages 2016

[18] MKaufmann ldquoUnstable proteinsHow to subject them to chro-matographic separations for purification proceduresrdquo Journalof Chromatography B Biomedical Sciences and Applications vol699 no 1-2 pp 347ndash369 1997

[19] T Geiger and S Clarke ldquoDeamidation isomerization andracemization at asparaginyl and aspartyl residues in peptidesSuccinimide-linked reactions that contribute to protein degra-dationrdquo The Journal of Biological Chemistry vol 262 no 2 pp785ndash794 1987

[20] L Wohlbrand H S Ruppersberg C Feenders B Blasius H-PBraun andR Rabus ldquoAnalysis ofmembrane-protein complexesof themarine sulfate reducerDesulfobacula toluolica Tol2 by 1Dblue native-PAGE complexome profiling and 2D blue native-SDS-PAGErdquo Proteomics vol 16 no 6 pp 973ndash988 2016

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samples (antibody enzyme DNA and diluted serum) weretreated with well-known reducing agents (ammonium fer-rous sulfate ascorbic acid sodiumhyposulfite and potassiumiodide) in the experimental group and with NaCl in thecontrol group The reducing agents (sodium hyposulfite andpotassium iodide) that had no inhibitory effect on biologicalmacromolecules were selected with using enzyme-linkedimmunosorbent assay (ELISA) and quantitative PCR (qPCR)to determine the activity of antibody enzyme and DNAAntibodies were treated with oxidants to cause oxidativedamage and then treated with reducing agents and thepossible repair of oxidative damage was assessed We believethat biological activity can be recovered following oxidativedamage using safe reducing agents which is the experimentalfoundation for antioxidant treatments in vitro andor in vivo

2 Materials and Methods

21 Effects of Reducing Agents on Antibody Activity

211 Reducing agents (6mmolL ammonium ferrous sulfate6mmolL ascorbic acid 60mmolL sodium hyposulfite and60mmolL potassium iodide) were diluted at a 11 ratio usingdistilled water to obtain eight concentrations NaCl solutionswere prepared with the same pH and ionic strength as thereductant solvent

212The samples (HBsAb healthy humanmixed serumwithan antibody to hepatitis B surface antigen collected froma hospital in Dalian China) were mixed with a reductantsolution or NaCl solution in a test tube at a ratio of 15 at 4∘Cfor 1 h

213 The antibody activity was detected with ELISA (hep-atitis B virus surface antibody test kit Chen Yu China) TheOD values were used to assess antibody activity with higherOD values indicating higher antibody activity The OD valuefor the blank was 0042 which is eliminated in the followingcalculation Inhibition rate = [(the OD value of the controlgroup ndash theODvalue of the blank hole) ndash (theODvalue of theexperimental group ndash the OD value of the blank hole)](theOD value of the control group ndash the OD value of the blankhole)times100 Data were analyzed using GraphPad Prism 5and are presented as the mean plusmn SD Statistical significancewas determined using Studentrsquos 119905-test and is presented aslowast(119901 lt 005) lowastlowast(119901 lt 001) or lowastlowastlowast(119901 lt 0001)

22 Effects of the Reducing Agents on Protein Structures

221Reducing agents were prepared at the following concen-trations 78mmolL ammonium ferrous sulfate 78mmolLascorbic acid 78mmolL sodium hyposulfite and 78mmolLpotassium iodide and NaCl solutions were prepared at theconcentrations of 78 and 78mmolL

222 The diluted serum (healthy human serum collectedfrom a hospital in Dalian China) was mixed with a reductantsolution or NaCl solution in a test tube at a ratio of 14 at 4∘Cfor 1 h and protected from light

223 Native-PAGE and SDS-PAGE were used to investigatewhether the spatial structures and molecular structuresof the proteins had changed [14ndash16] Separation gels andconcentrated gels were established at 12 and 5 Concen-trated gels were electrophoresed for 35min separation gelswere electrophoresed for 80min followed by staining withCoomassie Brilliant Blue and then decolorized

23 Effects of the Reducing Agents on the Activity ofTaq Enzyme and DNA

231 Reducing agents (10mmolL ammonium ferrous sul-fate 10mmolL ascorbic acid 100mmolL sodium hyposul-fite and 100mmolL potassium iodide) were diluted at a11 ratio using distilled water to obtain eight concentrationsNaCl solutions were prepared with the same pH and ionicstrength as the reductant solvent The Taq enzyme (SYBRPremix Ex Taq TMII Tli RNaseH Plus Takara Japan) wasmixed with a reductant solution or NaCl solution in a testtube at a ratio of 11 and the solutions were placed for 1 hand protected from light Other ingredients were not treatedexcept enzymes

232Reducing agents (6mmolL ammonium ferrous sulfate6mmolL ascorbic acid 60mmolL sodium hyposulfite and60mmolL potassium iodide) were diluted at a 11 ratiousing distilled water to obtain eight concentrations NaClsolutions were prepared with the same pH and ionic strengthas the reductant solvent DNA samples were extracted usinga DNA extraction kit (Omega Biotek Norcross GA USA)The primers for genes used in this study were szlig-actin (F51015840-CAAATATGAGATGCGTTGTTCAGG-31015840 R51015840-TGTGTG-GACTTGGGAGAGGA-31015840 ) and CRP (F51015840-AATGTGAAC-ATGTGGGACTTTGTG-31015840 R51015840-CGCCAGTTCAGGACA-TTAGGAC-31015840) The DNA was mixed with a reductantsolution orNaCl solution in a test tube at a ratio of 15 and thesolutions were placed for 1 h and protected from light Otheringredients were not treated except DNA

233 Ct values were determined using qPCR and corre-sponding Ct values for the same gene in the experimentalgroup and the control group were recorded Inhibition rateswere calculated using the following equation Inhibition rate= (Ct value of the experimental group ndash Ct value of thecontrol group)Ct value of the control group times100 Datawere analyzed using GraphPad Prism 5 and are presented asthe mean plusmn SD Statistical significance was determined usingStudentrsquos 119905-test and is presented as lowast(119901 lt 005) lowastlowast(119901 lt 001)or lowastlowastlowast(119901 lt 0001)

24 Possible Reversion of Oxidative Damage

241 Reductants with no significant inhibitory effects onthe activity of the antibody enzyme DNAand proteinstructures were selected via the procedures in Sections 2122 and 23 The antibody to HBsAg was treated withpotassium hypermanganate (25mmolL) to cause oxidativedamage [17] After removing the oxidant the antibody wastreated with reductant (50mmolL sodium hyposulfite and


Group A

Group B

Group C

Group D






HBsAb was treated

HBsAb was treated

HBsAb was treated

with NaCl

Wash

Wash

Wash

Wash


Processingof data

ELISAWash

Wash

with ＋2－Ｈ4

with ＋2－Ｈ4




3 Results

























4 Discussion













Group HBsAb 119901




Group HBsAb 119901










Data Availability




References
























Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Group A

Group B

Group C

Group D






HBsAb was treated

HBsAb was treated

HBsAb was treated

with NaCl

Wash

Wash

Wash

Wash


Processingof data

ELISAWash

Wash

with ＋2－Ｈ4

with ＋2－Ｈ4




3 Results

























4 Discussion













Group HBsAb 119901




Group HBsAb 119901










Data Availability




References
























Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018












4 Discussion













Group HBsAb 119901




Group HBsAb 119901










Data Availability




References
























Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018













Group HBsAb 119901




Group HBsAb 119901










Data Availability




References
























Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018









Data Availability




References
























Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





















Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


quantitativeassessment of the effects of reducing agents...

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