Auna tasirin methylene blue a mitokondriya: oxygen, ATP, da bambancin ƙarfin lantarkin membrane

A ɗauka cewa wani dakin gwaje-gwaje ya ƙara methylene blue ga mitokondriya da numfashinsu ya samu matsala, sannan ya ga cin oxygen ya ƙaru. Abin da za a fara tunanin rubutawa shi ne cewa rinin ya dawo da aikin mitokondriya. Wannan magana na iya zama ba daidai ba. Gudun oxygen, samar da ATP, da bambancin ƙarfin lantarkin membrane abubuwa ne uku mabambanta. Suna amfani da gaɓar ƙwayar halitta guda ɗaya kamar yadda ajiyar kwafi, daidaita fayiloli, da rabawa suke amfani da babban fayil ɗaya a girgije: suna da alaƙa, amma ƙa'idoji dabam suke sarrafa su. Idan aka ɗauki ɗaya a matsayin wani, fassarar sakamakon za ta gaza, ko da duk ma'aunan an yi su daidai a fasahance.

Methylene blue yana ƙara yiwuwar wannan ruɗani saboda yana shiga cikin tsarin da ake aunawa. Yana zagayawa tsakanin karɓa da bayar da electron a tsarin redox, yana karɓar electron daga matakan farko ya ba da su ga matakan gaba; cation ne mai son kitse wanda yake taruwa a mitokondriya masu kuzari; kuma sinadari ne mai tsananin launin shuɗi wanda yake sha da fitar da haske a zangon da yawancin abubuwan ganowa suke amfani da shi. Sakamakon ma'auni guda ɗaya ba zai iya ɗaukar wannan nauyi ba. Tsarin da ake iya dogaro da shi, wanda binciken mitokondriya na Budapest ya nuna, shi ne auna gudun oxygen tare da saurin samar da ATP, bambancin ƙarfin lantarkin membrane, da fitowar hydrogen peroxide, tare da gwaje-gwajen tantancewa na musamman ga rinin a kowane mataki.

Gwajin numfashi yana auna gudun oxygen, ba yawan kuzarin da ake samarwa ba

Gwajin numfashi mai tsananin iya bambance ƙananan sauye-sauye, ko da Oroboros Oxygraph ne ko na'urar Seahorse mai nazarin gudun sinadarai a wajen ƙwayoyin halitta, yana bayar da bayani guda ɗaya: saurin karɓar electron da oxygen yake yi. Ba ya bayyana adadin proton da aka tura ga kowane electron, yawan bambancin matakin da aka adana, ko ko ATP synthase ya yi amfani da shi.

Kalmomin da ake amfani da su suna da muhimmanci. A mitokondriya da aka ware, sinadarin da ake sarrafawa ba tare da ADP ba yana bayar da numfashin zubewar proton, sinadarin tare da ADP mai isasshen yawa yana bayar da ƙarfin samar da ATP ta hanyar oxidative phosphorylation, sannan ƙara sinadarin raba haɗin numfashi da samar da ATP a hankali yana bayar da iyakar ƙarfin canja wurin electron. A ƙwayoyin halitta masu cikakken tsari, makamantan ma'aunan Seahorse su ne numfashi na asali, numfashi mai alaƙa da ATP wanda oligomycin yake ragewa, zubewar proton, iyakar numfashi da FCCP yake ƙayyadewa, da cin oxygen wanda ba na mitokondriya ba kuma rotenone da antimycin ba sa dakatar da shi. Kiran sakamakon Seahorse State 3 da State 4 yana ɗora masa daidaiton da samfurin bai ba da damar samu ba, kamar yadda tsarin Agilent Mito Stress Test ya bayyana.

Methylene blue yana karya alaƙar da ake tsammani tsakanin ƙarin oxygen da ƙarin kuzari. A mitokondriyar kwakwalwar guinea pig da aka ware, rini daga 100 nM zuwa 1 uM ya ƙara cin oxygen a lokacin hutawa da sinadaran glutamate tare da malate, succinate, da alpha-glycerophosphate; ƙarin ya kai kusan kashi 64 bisa ɗari da alpha-glycerophosphate. Duk da haka, numfashin da ADP ya ƙarfafa a mitokondriya masu lafiya bai canja sosai ba, kamar yadda binciken mitokondriyar kwakwalwa da aka ware ya nuna. Saboda numfashin zubewar proton ya ƙaru yayin da gudun da ADP ya ƙarfafa ya tsaya daidai, rabon sarrafa numfashi da aka lissafa yana raguwa. Wannan raguwar ba ta nufin rinin ya lalata mitokondriya ba. Tana nufin rinin ya ƙara wani zagayen redox mai cin oxygen a kan numfashi na al'ada. Gwajin cin oxygen na methylene blue wanda yake bayar da rahoton ƙarin kawai ya auna wani aiki na sinadarai na gaske, amma bai faɗi komai game da adana kuzari ba.

Gwaje-gwajen dawo da aiki suna nuna wannan gibin a ma'auni mafi girma. Lokacin da aka toshe Complex I, gudun oxygen ya dawo zuwa kusan ninki uku, amma samar da ATP ya dawo zuwa kusan ninki 1.5 kawai. Ƙarin oxygen ya isa electrode. Ƙaramin kaso daga cikinsa ya samar da kuzarin ATP. Duk wata magana cewa oxidative phosphorylation ya inganta tana buƙatar bayanan ATP, kuma ya fi kyau a bayyana su a matsayin rabon ATP ga oxygen tare da bayyana ƙa'idar abin da aka sa a ƙasan rabon.

Gwaje-gwajen ATP suna auna saurin samarwa, wanda yawan ATP da ake da shi ba zai iya maye gurbinsa ba

Gwajin ATP yana amsa wata tambaya dabam: nawa ne ATP ake samarwa a cikin kowace raka'ar lokaci. Ma'aunin da ake bin sauyawarsa da lokaci, ko hasken luciferase na ƙwaron fitila da ake bi na mintuna, ko gwajin haɗin hexokinase da glucose-6-phosphate dehydrogenase da ake karantawa a 340 nm, yana bayar da sauri. Auna jimillar ATP sau ɗaya a ƙarshen gwaji yana bayar da yawan da yake akwai, wanda yake nuna samarwa bayan an cire abin da aka cinye, kuma shi kaɗai ba ya bayar da cikakken bayani game da oxidative phosphorylation.

Ƙungiyar Budapest ta yi amfani da hanyar haɗin enzymes tare da hana aikin adenylate kinase, kuma ta gano cewa methylene blue a 1 zuwa 2 uM ya ƙara samar da ATP kaɗan amma da muhimmanci a kididdiga, a mitokondriya da aka yi wa rotenone da waɗanda aka yi wa antimycin. Wannan dawo da aiki ne na gaske, amma na wani ɓangare kawai. Binciken gubar TEGDMA ya samu amsa mai irin wannan tsari a wani samfurin gwaji dabam: 5 mM na monomer ɗin hakori TEGDMA ya rage numfashin da yake da alaƙa da Complex I daga kusan 286 zuwa 28 na raka'o'in gudu, sannan 2 uM na rinin ya dawo da shi zuwa kusan 86.

Akwai gwaje-gwajen tantancewa biyu da dole ne a yi da sinadari mai launi wanda yake shiga redox. Na farko, a yi amfani da oligomycin domin tabbatar da nawa daga samar da ATP da aka auna ya dogara da ATP synthase. Na biyu, a yi gwajin gano ƙarin ATP da aka sa a samfurin da ba shi da ƙwayoyin halitta, tare da adadin ATP da aka sani, cikakken sinadarin gwaji, da ainihin matakan yawan rinin da aka yi amfani da su; aƙalla 0, 100 nM, 300 nM, 1 uM, da 2 uM. Rinin zai iya shan hasken da aka fitar, hana aikin enzyme mai bayar da sigina, ko canja sinadarin redox na kayan gwajin. Jagorar gwajin luciferase ta gama-gari tana ba da shawarar gwaje-gwajen sinadarai na tantance tsoma baki saboda irin wannan matsala. Kada a ɗauka cewa 1 uM ba ya haifar da karkatar sakamako saboda kashe haske yawanci ya fi tsanani a matakan yawa mafi girma.

Abubuwan gano bambancin ƙarfin lantarkin membrane suna auna rarrabuwar rini, ba gudunsa ba

Safranine O, TMRM da TMRE, rhodamine 123, da JC-1 duk cations ne masu iya ratsa membrane. Suna taruwa a cikin matrix mai cajin korau bisa daidaiton lantarki da sinadarai, don haka jimillar fluorescence tana nuna bambancin ƙarfin lantarki a kaikaice ta wurin da abin ganowa ya zauna. Babu ɗayansu da yake auna ATP ko gudun electron.

Kowane abin ganowa yana da nau'in samfurin da ya fi dacewa da shi. Safranine O ya dace da mitokondriya da aka ware da ƙwayoyin halitta da aka buɗe membrane ɗinsu domin sinadarai su ratsa, yawanci a kusan 2 uM, da hasken tayarwa a kusan 495 nm da hasken fitowa a kusan 585 nm. Yana aiki a yanayin kashe haske, inda shigarsa cikin mitokondriya yake canja jimillar fluorescence. TMRM da TMRE sun dace da ƙwayoyin halitta masu cikakken tsari idan an sa su da ƙananan matakan nanomolar a yanayin da ba ya kashe haske; a nan, raguwar bambancin ƙarfin lantarki tana sakin rini, kuma fluorescence na mitokondriya yana raguwa. Idan an sa rini da yawa, siginar na iya sauya akalarta saboda rinin yana kashe haskensa da kansa. JC-1 yana bayar da rabon kore ga ja mai sauƙin amfani, amma ya dogara da yawan rinin da aka sa da yawan mitokondriya, don haka bai kai TMRM da aka sarrafa da kulawa dacewa da aikin auna adadi ba. An taƙaita waɗannan fa'idodi da gazawa a wani bita kan abubuwan gano bambancin ƙarfin lantarkin membrane na mitokondriya da ake yawan amfani da shi.

Methylene blue yana tsoma baki a ma'aunin haske a ainihin matakan yawan da ake amfani da su. Zangonsa na shan haske a kusan 600 zuwa 665 nm yana haɗuwa da hasken ja da abubuwan ganowa suke fitarwa, don haka tasirin tace haske a cikin samfurin, kashe haske, da shan hasken tayarwa duk suna iya faruwa. Rinin kuma cation ne na biyu mai son kitse, wanda rarrabuwarsa take canjawa da bambancin ƙarfin lantarki da ake aunawa. Hanyar kariya ita ce amfani da ma'aunai fiye da ɗaya tare da samfuran gwaji marasa abin aunawa: abin ganowa kaɗai, rini kaɗai, abin ganowa tare da kowane matakin yawan rini, kawar da bambancin ƙarfin lantarki gaba ɗaya da FCCP a matsayin ma'aunin sifili, kuma idan zai yiwu wani abin ganowa na biyu mai zangon haske dabam. Binciken kauce wa Complex III ya kafa wannan misali ta hanyar maimaita gwajin dawo da bambancin ƙarfin lantarkin membrane da safranine O da TMRM bayan antimycin da myxothiazol sun hana aiki, kuma an samu dawo da aiki iri ɗaya a yanayinsa. Abin bayar da siginar haske guda ɗaya yana ba da alama. Masu bayar da sigina biyu masu zaman kansu suna bayar da hujja.

Samfura uku, tambayoyi uku mabambanta

Mitokondriya da aka ware, ƙwayoyin halitta da aka buɗe membrane ɗinsu domin sinadarai su ratsa, da ƙwayoyin halitta masu cikakken tsari ba matakan daidaita ƙarfin gano sauyi na gwaji guda ɗaya ba ne da za a iya musanya su. Tsare-tsare ne mabambanta masu ƙa'idojin aiki dabam.

Mitokondriya da aka ware suna riƙe da sarkar jigilar electron, membrane na ciki, da tsarin aikin matrix, amma an cire membrane na ƙwayar halitta, cytosol, da tsarin hanyar haɗin mitokondriya. Wannan cirewar ita ce manufar: ana ƙayyade sinadaran da za a sarrafa, ADP, abubuwan hana aiki, da matakan yawan rini daidai, don haka ana iya tantance wata hanya. Abin da ake rasa shi ne samar da sinadaran da ake sarrafawa ta hanyar halitta, isar da sigina, da buƙatar kuzari.

Ƙwayoyin halitta da aka buɗe membrane ɗinsu suna riƙe da mitokondriya a cikin babban ɓangaren tsarin ƙwayar halittar, amma suna barin sinadaran da aka kawo daga waje da ADP su ratsa membrane na ƙwayar halitta da aka buɗe. Su ne kayan aikin da suka dace wajen tantance matsalar hanyar sarrafa wani sinadari a cikin tsarin ƙwayar halitta. Buɗewa fiye da kima yana lalata membrane na wajen mitokondriya, kuma tsarin sarrafawa na cytosol yana wankewa ya fita.

Ƙwayoyin halitta masu cikakken tsari suna riƙe da komai: shingen membrane na ƙwayar halitta, sinadaran bayar da electron a cytosol, jigilar sinadaran da ake sarrafawa daga cikin jiki, hanyar haɗin mitokondriya, da buƙatar ATP. Su ne mafi kusa da yanayin aikin halitta kuma mafi nisa da bayyana hanyar aikin sinadari, domin shigar rini, karɓar electron ɗinsa, da kulle shi a wasu wurare duk suna ƙayyade yawan da yake kai wa mitokondriya a zahiri.

Binciken TEGDMA ya yi amfani da waɗannan matakai uku da gangan: mitokondriyar kwakwalwa da aka ware domin dawo da oxidation da bambancin ƙarfin lantarki ta hanyar rini; ƙwayoyin tushe na cikin hakori da aka buɗe membrane ɗinsu, musamman domin gano cewa matsalar tana a oxidation na sinadaran da yake da alaƙa da Complex I; da ƙwayoyin tushe masu cikakken tsari domin auna jimillar tasirin 2 uM na rini a yanayin aikin halitta kan cin oxygen da aka rage. Ga masu karatu da suke kwatanta ma'aunan mitokondriya tsakanin samfura, bayanin haɗin gwiwa kan yadda methylene blue yake motsa electron ta sarkar numfashi yana bayar da tushen hanyoyin, yayin da jagorar karanta binciken numfashi na methylene blue take bayyana dalilin zaɓen adadin sinadari da abubuwan hana aiki.

Gwaje-gwajen tantancewa da rini mai shiga redox yake buƙata

Sinadarin da yake zagayawa tsakanin karɓa da bayar da electron yana buƙatar jerin gwaje-gwajen tantancewa mafi tsawo fiye da abin hana aiki na yau da kullum, domin aikin sinadarin rini shi kaɗai zai iya kwaikwayon aikin halitta.

Don gwajin numfashi, a yi gwaji a ɗaki mai ruwan buffer kaɗai domin auna karkacewar ma'auni, a ɗaki mai buffer da rini ba tare da kayan halitta ba, da rini tare da duk wani sinadarin bayar da electron a wajen ƙwayoyin halitta da yake cikin gwajin. A kuma yi amfani da rotenone da antimycin a ƙarshen gwaji domin ƙayyade cin oxygen wanda ba na mitokondriya ba a ƙwayoyin halitta masu cikakken tsari. A lura cewa rinin da ya karɓi electron yana sake ba da su ga oxygen, yana samar da hydrogen peroxide. Saboda haka, samfurin tantancewa mai rini kaɗai ba tare da mai bayar da electron ba yana ƙiyasta cin oxygen ba tare da ƙwayoyin halitta ba ƙasa da na gaske, wanda yake bayyana da zarar NADH ko NADPH ya samu.

Don hydrogen peroxide, a fi son Amplex UltraRed da aka daidaita ma'auninsa tare da horseradish peroxidase maimakon rinayen DCFDA marasa keɓantaccen zaɓi. A tabbatar da sakamakon da catalase, kuma a gwada ko ya dogara da haske, domin rinin yana ƙara yiwuwar aikin sinadarai idan haske ya same shi. Binciken mitokondriyar kwakwalwa ya gano cewa yawan peroxide ya ƙaru fiye da ninki huɗu da rini na 100 nM kawai, yayin da kawar da shi ta ragu. Rinin da ya karɓi electron ne ya samar da peroxide kai tsaye, ba tare da superoxide a matsayin matakin tsaka-tsaki ba. Cikakken bayani kan zaɓen abin ganowa da gwaje-gwajen tantance tasirin haske yana cikin maƙalar haɗin gwiwa kan auna nau'ikan oxygen masu saurin yin hulɗar sinadarai a gwaje-gwajen rini. An kuma tattauna tambaya mafi faɗi ta lokacin da peroxide yake aiki a matsayin sigina ko nauyi tare da daidaiton redox a ƙwayoyin halitta da maganganun antioxidants.

Don maganganu kan hanyar aiki, a gwada wurare biyu na hana aikin Complex III: antimycin A a wurin Qi da myxothiazol a wurin Qo, sannan a gwada Complex IV a matakin ƙarshe da cyanide. Wani binciken kwakwalwar bera na baya ya bayar da rahoton cewa rinin ya dawo da numfashin da rotenone ya toshe, amma bai dawo da wanda antimycin ya toshe ba. Sai dai binciken daga baya a nau'ikan halittu uku ya samu dawo da wani ɓangaren aiki da duka abubuwan hana aikin, tare da hujjar karɓar electron kai tsaye ta cytochrome c. A ɗauki hanyar kaucewar a matsayin wadda ta dace da sabbin bayanai, ba abin da aka tabbatar gaba ɗaya ba, domin yawan rini, wurin hana aiki, yanayin sinadarin da ake sarrafawa, da rarrabuwar rini duk suna canja hanyar da ake gani.

Teburin zaɓen gwaji

A yi amfani da wannan tebur domin zaɓen ma'auni bisa tambayar da ake son amsawa, ba zaɓen tambaya bisa ma'aunin da ake da shi ba.

Mafi ƙarancin abin da zai sa magana ta zama mai gamsarwa shi ne gwada matakan yawan rini iri ɗaya, 100 nM, 300 nM, 1 uM, da 2 uM, wajen auna gudun oxygen a yanayin zubewar proton, ADP, oligomycin, da sinadarin raba haɗin numfashi da samar da ATP; saurin samar da ATP; bambancin ƙarfin lantarkin membrane, zai fi kyau da abubuwan ganowa biyu; da fitowar peroxide da aka daidaita ma'auninta, tare da catalase da gwaje-gwajen tantancewa marasa ƙwayoyin halitta. A bayar da rahoton ma'aunan da suke da alaƙa: gudun oxygen tare da saurin ATP, ATP ga oxygen, bambancin ƙarfin lantarki, da peroxide. Kowane ɗaya daga cikin waɗannan lambobi shi kaɗai ma'auni ne da har yanzu yake neman ma'ana.