Native mass spectrometry of human carbonic anhydrase I and its inhibitor complexes

Carlotta Zoppi1, Alessio Nocentini2, Alessandro Pratesi3, Luigi Messori4
1Laboratory of Metals in Medicine (MetMed), Department of Chemistry Ugo Schiff, University of Florence, Via della Lastruccia 3-13, 50019 Sesto Fiorentino, Italy.
2Department of Neurofarba, Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Via U. Schiff 6, 50019, Sesto Fiorentino, Italy.
3Department of Chemistry and Industrial Chemistry, University of Pisa, Via G. Moruzzi 13, 56124 Pisa, Italy
4Laboratory of Metals in Medicine (MetMed), Department of Chemistry Ugo Schiff, University of Florence, Sesto Fiorentino, Italy

Tóm tắt

Abstract

Native mass spectrometry is a potent technique to study and characterize biomacromolecules in their native state. Here, we have applied this method to explore the solution chemistry of human carbonic anhydrase I (hCA I) and its interactions with four different inhibitors, namely three sulfonamide inhibitors (AAZ, MZA, SLC-0111) and the dithiocarbamate derivative of morpholine (DTC). Through high-resolution ESI-Q-TOF measurements, the native state of hCA I and the binding of the above inhibitors were characterized in the molecular detail. Native mass spectrometry was also exploited to assess the direct competition in solution among the various inhibitors in relation to their affinity constants. Additional studies were conducted on the interaction of hCA I with the metallodrug auranofin, under various solution and instrumental conditions. Auranofin is a selective reagent for solvent-accessible free cysteine residues, and its reactivity was analyzed also in the presence of CA inhibitors. Overall, our investigation reveals that native mass spectrometry represents an excellent tool to characterize the solution behavior of carbonic anhydrase.

Graphic abstract

Từ khóa


Tài liệu tham khảo

Fegler J (1944) Function of carbonic anhydrase in blood. Nature 153:137–138

Lindskog S (1997) Structure and mechanism of carbonic anhydrase. Pharmacol Ther 74:1–20

Pocker Y, Tanaka N (1978) Inhibition of carbonic anhydrase by anions in the carbon dioxide-bicarbonate system. Science (80-) 199:907–909. https://doi.org/10.1126/science.199.4331.907

Mann T, Keilin D (1940) Sulphanilamide as a specific inhibitor of carbonic anhydrase. Nature 146:164–165

Supuran CT (2008) Carbonic anhydrases: Novel therapeutic applications for inhibitors and activators. Nat Rev Drug Discov 7:168–181

Supuran CT (2016) Carbonic anhydrase inhibition and the management of neuropathic pain. Expert Rev Neurother 16:961–968

Supuran CT, Scozzafava A, Casini A (2003) Carbonic anhydrase inhibitors. Med Res Rev 23:146–189. https://doi.org/10.1002/med.10025

Supuran CT, Scozzafava A (2000) Carbonic anhydrase inhibitors: aromatic sulfonamides and disulfonamides act as efficient tumor growth inhibitors. J Enzyme Inhib 15:597–610. https://doi.org/10.3109/14756360009040713

Ferraroni M, Cornelio B, Sapi J, Supuran CT, Scozzafava A (2018) Sulfonamide carbonic anhydrase inhibitors: zinc coordination and tail effects influence inhibitory efficacy and selectivity for different isoforms. Inorganica Chim Acta 470:128–132. https://doi.org/10.1016/j.ica.2017.03.038

Degn H, Kristensen B (1986) Stopped flow mass spectrometry: applications to the carbonic anhydrase reaction. J Biochem Biophys Methods 12:305–310. https://doi.org/10.1016/0165-022X(86)90067-9

Day YSN, Baird CL, Rich RL, Myszka DG (2002) Direct comparison of binding equilibrium, thermodynamic, and rate constants determined by surface- and solution-based biophysical methods. Protein Sci 11:1017–1025. https://doi.org/10.1110/ps.4330102

Krishnamurthy VM, Kaufman GK, Urbach AR, Gitlin I, Gudiksen KL, Weibel DB, Whitesides GM (2008) Carbonic anhydrase as a model for biophysical and physical-organic studies of proteins and protein-ligand binding. Chem Rev 108:946–1051

Pacholarz KJ, Garlish RA, Taylor RJ, Barran PE (2012) Mass spectrometry based tools to investigate protein–ligand interactions for drug discovery. Chem Soc Rev 41:4335. https://doi.org/10.1039/c2cs35035a

Siegel M (2002) Early discovery drug screening using mass spectrometry. Curr Top Med Chem 2:13–33

Lee M (2005) Integrated strategies for drug discovery using mass spectrometry. Wiley, Hoboken

Hofstadler SA, Sannes-Lowery KA (2006) Applications of ESI-MS in drug discovery: interrogation of noncovalent complexes. Nat Rev Drug Discov 5:585–595

Leney AC, Heck AJR (2017) Native mass spectrometry: what is in the Name? J Am Soc Mass Spectrom 28:5–13. https://doi.org/10.1007/s13361-016-1545-3

Breuker K, McLafferty FW (2008) Stepwise evolution of protein native structure with electrospray into the gas phase, 10–12 to 102 s. Proc Natl Acad Sci USA 105:18145–18152

Ganem B, Li YT, Henion JD (1991) Detection of noncovalent receptor-ligand complexes by mass spectrometry. J Am Chem Soc 113:6294–6296. https://doi.org/10.1021/ja00016a069

Katta V, Chait BT (1991) Observation of the heme–globin complex in native myoglobin by electrospray-ionization mass spectrometry. J Am Chem Soc 113:8534–8535. https://doi.org/10.1021/ja00022a058

Loo JA (1997) Studying noncovalent protein complexes by electrospray ionization mass spectrometry. Mass Spectrom Rev 16:1–23. https://doi.org/10.1002/(SICI)1098-2787(1997)16:1<1:AID-MAS1>3.0.CO;2-L

Hofstadler SA, Griffey RH (2001) Analysis of noncovalent complexes of DNA and RNA by mass spectrometry. Chem Rev 101:377–390. https://doi.org/10.1021/cr990105o

Woods LA, Dolezal O, Ren B, Ryan JH, Peat TS, Poulsen SA (2016) Native state mass spectrometry, surface plasmon resonance, and X-ray crystallography correlate strongly as a fragment screening combination. J Med Chem 59:2192–2204. https://doi.org/10.1021/acs.jmedchem.5b01940

Chowdhury SK, Katta V, Chait BT (1990) Probing conformational changes in proteins by mass spectrometry. J Am Chem Soc 112:9012–9013. https://doi.org/10.1021/ja00180a074

Kaltashov IA, Zhang M, Eyles SJ, Abzalimov RR (2006) Investigation of structure, dynamics and function of metalloproteins with electrospray ionization mass spectrometry. Anal Bioanal Chem 386:472–481

Kaltashov AI, Mohimen A (2005) Estimates of protein surface areas in solution by electrospray ionization mass spectrometry. Anal Chem 77:5370–5379. https://doi.org/10.1021/ac050511+

Grandori R, Santambrogio C, Brocca S, Invernizzi G, Lotti M (2009) Electrospray-ionization mass spectrometry as a tool for fast screening of protein structural properties. Biotechnol J 4:73–87. https://doi.org/10.1002/biot.200800250

Li J, Santambrogio C, Brocca S, Rossetti G, Carloni P, Grandori R (2016) Conformational effects in protein electrospray-ionization mass spectrometry. Mass Spectrom Rev 35:111–122. https://doi.org/10.1002/mas.21465

Konermann L, Douglas DJ (1997) Acid-induced unfolding of cytochrome c at different methanol concentrations: electrospray ionization mass spectrometry specifically monitors changes in the tertiary structure. Biochemistry 36:12296–12302. https://doi.org/10.1021/bi971266u

Nabuchi Y, Murao N, Asoh Y, Takayama M (2007) Probing the unfolding and refolding processes of carbonic anhydrase 2 using electrospray ionization mass spectrometry combined with pH jump. Anal Chem 79:8342–8349. https://doi.org/10.1021/ac071130u

Carta F, Aggarwal M, Maresca A, Scozzafava A, McKenna R, Masini E, Supuran CT (2012) Dithiocarbamates strongly inhibit carbonic anhydrases and show antiglaucoma action in vivo. J Med Chem 55:1721–1730. https://doi.org/10.1021/jm300031j

Pacchiano F, Aggarwal M, Avvaru BS, Robbins AH, Scozzafava A, McKenna R, Supuran CT (2010) Selective hydrophobic pocket binding observed within the carbonic anhydrase II active site accommodate different 4-substituted-ureido-benzenesulfonamides and correlate to inhibitor potency. Chem Commun 46:8371–8373. https://doi.org/10.1039/c0cc02707c

Aggarwal M, Kovalevsky AY, Velazquez H, Fisher SZ, Smith JC, McKenna R (2016) Neutron structure of human carbonic anhydrase II in complex with methazolamide: mapping the solvent and hydrogen-bonding patterns of an effective clinical drug. IUCrJ 3:319–325. https://doi.org/10.1107/S2052252516010514

Sippel KH, Robbins AH, Domsic J, Genis C, Agbandje-Mckenna M, McKenna R (2009) High-resolution structure of human carbonic anhydrase II complexed with acetazolamide reveals insights into inhibitor drug design. Acta Crystallogr Sect F Struct Biol Cryst Commun 65:992–995. https://doi.org/10.1107/S1744309109036665

McDonald PC, Chia S, Bedard PL, Chu Q, Lyle M, Tang L, Singh M, Zhang Z, Supuran CT, Renouf DJ, Dedhar S (2020) A phase 1 study of SLC-0111, a novel inhibitor of carbonic anhydrase IX, in patients with advanced solid tumors. Am J Clin Oncol Cancer Clin Trials 43:484–490. https://doi.org/10.1097/COC.0000000000000691

Nocentini A, Supuran CT (2019) Advances in the structural annotation of human carbonic anhydrases and impact on future drug discovery. Expert Opin Drug Discov 14:1175–1197

Avvaru BS, Kim CU, Sippel KH, Gruner SM, Agbandje-McKenna M, Silverman DN, McKenna R (2010) A short, strong hydrogen bond in the active site of human carbonic anhydrase II. Biochemistry 49:249–251. https://doi.org/10.1021/bi902007b

Zoppi C, Messori L, Pratesi A (2020) ESI MS studies highlight the selective interaction of Auranofin with protein free thiols. Dalt Trans 49:5906–5913. https://doi.org/10.1039/d0dt00283f

King RW, Roberts GCK (1971) Nuclear magnetic resonance studies of human carbonic anhydrase B. Histidine residues. Biochemistry 10:558–565. https://doi.org/10.1021/bi00780a003

Song H, Wilson DL, Farquhar ER, Lewis EA, Emerson JP (2012) Revisiting zinc coordination in human carbonic anhydrase II. Inorg Chem 51:11098–11105. https://doi.org/10.1021/ic301645j

Brasuń J, Matera-Witkiewicz A, Ołdziej S, Pratesi A, Ginanneschi M, Messori L (2009) Impact of ring size on the copper(II) coordination abilities of cyclic tetrapeptides. J Inorg Biochem 103:813–817. https://doi.org/10.1016/j.jinorgbio.2009.02.003

Matera-Witkiewicz A, Brasuń J, Światek-Kozłowska J, Pratesi A, Ginanneschi M, Messori L (2009) Short-chain oligopeptides with copper(II) binding properties: the impact of specific structural modifications on the copper(II) coordination abilities. J Inorg Biochem 103:678–688. https://doi.org/10.1016/j.jinorgbio.2009.01.001

Coleman JE (1965) Human carbonic anhydrase. Protein conformation and metal ion binding. Biochemistry 4:2644–2655. https://doi.org/10.1021/bi00888a014

Pratesi A, Cirri D, Ciofi L, Messori L (2018) Reactions of auranofin and its pseudohalide derivatives with serum albumin investigated through ESI-Q-TOF MS. Inorg Chem 57:10507–10510. https://doi.org/10.1021/acs.inorgchem.8b02177

Michelucci E, Pieraccini G, Moneti G, Gabbiani C, Pratesi A, Messori L (2017) Mass spectrometry and metallomics: a general protocol to assess stability of metallodrug-protein adducts in bottom-up MS experiments. Talanta 167:30–38. https://doi.org/10.1016/j.talanta.2017.01.074

Marzo T, De Pascali SA, Gabbiani C, Fanizzi FP, Messori L, Pratesi A (2017) ESI–MS studies of the reactions of novel platinum(II) complexes containing O,O′-chelated acetylacetonate and sulfur ligands with selected model proteins. Biometals 30:609–614. https://doi.org/10.1007/s10534-017-0031-0

Pratesi A, Cirri D, Đurović MD, Pillozzi S, Petroni G, Bugarčić ŽD, Messori L (2016) New gold carbene complexes as candidate anticancer agents. Biometals 29:905–911. https://doi.org/10.1007/s10534-016-9962-0

Massai L, Pratesi A, Bogojeski J, Banchini M, Pillozzi S, Messori L, Bugarčić ŽD (2016) Antiproliferative properties and biomolecular interactions of three Pd(II) and Pt(II) complexes. J Inorg Biochem 165:1–6. https://doi.org/10.1016/j.jinorgbio.2016.09.016

Tamasi G, Carpini A, Valensin D, Messori L, Pratesi A, Scaletti F, Jakupec M, Keppler B, Cini R (2014) {Ru(CO)x}-core complexes with selected azoles: synthesis, X-ray structure, spectroscopy, DFT analysis and evaluation of cytotoxic activity against human cancer cells. Polyhedron 81:227–237

Heck AJR (2008) Native mass spectrometry: a bridge between interactomics and structural biology. Nat Methods 5:927–933. https://doi.org/10.1038/nmeth.1265

Donnelly DP, Rawlins CM, DeHart CJ, Fornelli L, Schachner LF, Lin Z, Lippens JL, Aluri KC, Sarin R, Chen B, Lantz C, Jung W, Johnson KR, Koller A, Wolff JJ, Campuzano IDG, Auclair JR, Ivanov AR, Whitelegge JP et al (2019) Best practices and benchmarks for intact protein analysis for top-down mass spectrometry. Nat Methods 16:587–594. https://doi.org/10.1038/s41592-019-0457-0

Yin S, Loo JA (2011) Top-down mass spectrometry of supercharged native protein-ligand complexes. Int J Mass Spectrom 300:118–122. https://doi.org/10.1016/j.ijms.2010.06.032

Lomeli SH, Yin S, Ogorzalek Loo RR, Loo JA (2009) Increasing charge while preserving noncovalent protein complexes for ESI-MS. J Am Soc Mass Spectrom 20:593–596. https://doi.org/10.1016/j.jasms.2008.11.013

Natalello A, Santambrogio C, Grandori R (2017) Are charge-state distributions a reliable tool describing molecular ensembles of intrinsically disordered proteins by native MS? J Am Soc Mass Spectrom 28:21–28. https://doi.org/10.1007/s13361-016-1490-1

Hamdy OM, Julian RR (2012) Reflections on charge state distributions, protein structure, and the mystical mechanism of electrospray ionization. J Am Soc Mass Spectrom 23:1–6. https://doi.org/10.1007/s13361-011-0284-8

Kafader JO, Melani RD, Schachner LF, Ives AN, Patrie SM, Kelleher NL, Compton PD (2020) Native vs denatured: an in depth investigation of charge state and isotope distributions. J Am Soc Mass Spectrom 31:574–581. https://doi.org/10.1021/jasms.9b00040

Heck AJR, van den Heuvel RHH (2004) Investigation of intact protein complexes by mass spectrometry. Mass Spectrom Rev 23:368–389. https://doi.org/10.1002/mas.10081

Yan X, Watson J, Ho PS, Deinzer ML (2004) Mass spectrometric approaches using electrospray ionization charge states and hydrogen-deuterium exchange for determining protein structures and their conformational changes. Mol Cell Proteom 3:10–23

Krusemark CJ, Frey BL, Belshaw PJ, Smith LM (2009) Modifying the charge state distribution of proteins in electrospray ionization mass spectrometry by chemical derivatization. J Am Soc Mass Spectrom 20:1617–1625. https://doi.org/10.1016/j.jasms.2009.04.017

Menchise V, De SG, Di FA, Scozzafava A, Supuran CT (2006) Carbonic anhydrase inhibitors: X-ray crystallographic studies for the binding of 5-amino-1,3,4-thiadiazole-2-sulfonamide and 5-(4-amino-3-chloro-5-fluorophenylsulfonamido)-1,3,4-thiadiazole-2-sulfonamide to human isoform II. Bioorganic Med Chem Lett 16:6204–6208. https://doi.org/10.1016/j.bmcl.2006.09.022

Alterio V, Di Fiore A, D’Ambrosio K, Supuran CT, De Simone G (2012) Multiple binding modes of inhibitors to carbonic anhydrases: How to design specific drugs targeting 15 different isoforms? Chem Rev 112:4421–4468

Winum JY, Supuran CT (2015) Recent advances in the discovery of zinc-binding motifs for the development of carbonic anhydrase inhibitors. J Enzyme Inhib Med Chem 30:321–324

Gao J, Wu Q, Carbeck J, Lei QP, Smith RD, Whitesides GM (1999) Probing the energetics of dissociation of carbonic anhydrase-ligand complexes in the gas phase. Biophys J 76:3253–3260. https://doi.org/10.1016/S0006-3495(99)77477-1

Dugad LB, Cooley CR, Gerig JT (1989) NMR studies of carbonic anhydrase-fluorinated benzenesulfonamide complexes. Biochemistry 28:3955–3960. https://doi.org/10.1021/bi00435a049

Kovalevsky A, Aggarwal M, Velazquez H, Cuneo MJ, Blakeley MP, Weiss KL, Smith JC, Fisher SZ, McKenna R (2018) “To be or not to be” protonated: atomic details of human carbonic anhydrase-clinical drug complexes by neutron crystallography and simulation. Structure 26:383–390.e3. https://doi.org/10.1016/j.str.2018.01.006

Avvaru BS, Busby SA, Chalmers MJ, Griffin PR, Venkatakrishnan B, Agbandje-McKenna M, Silverman DN, McKenna R (2009) Apo-human carbonic anhydrase II revisited: implications of the loss of a metal in protein structure, stability, and solvent network. Biochemistry 48:7365–7372. https://doi.org/10.1021/bi9007512

Ray SS, Singh SK, Balaram P (2001) An electrospray ionization mass spectrometry investigation of 1-anilino-8-naphthalene-sulfonate (ANS) binding to proteins. J Am Soc Mass Spectrom 12:428–438. https://doi.org/10.1016/S1044-0305(01)00206-9

Thomson BA (1997) Declustering and fragmentation of protein ions from an electrospray ion source. J Am Soc Mass Spectrom 8:1053–1058. https://doi.org/10.1016/S1044-0305(97)00129-3

Susa AC, Xia Z, Tang HYH, Tainer JA, Williams ER (2017) Charging of proteins in native mass spectrometry. J Am Soc Mass Spectrom 28:332–340. https://doi.org/10.1007/s13361-016-1517-7

Huang EC, Pramanik BN, Tsarbopoulos A, Reichert P, Ganguly AK, Trotta PP, Nagabhushan TL, Covey TR (1993) Application of electrospray mass spectrometry in probing protein-protein and protein-ligand noncovalent interactions. J Am Soc Mass Spectrom 4:624–630. https://doi.org/10.1016/1044-0305(93)85026-T

Baca M, Kent SBH (1992) Direct observation of a ternary complex between the dimeric enzyme HIV-1 protease and a substrate-based inhibitor. J Am Chem Soc 114:3992–3993

Busemann A, Araman C, Flaspohler I, Pratesi A, Zhou X-Q, van Rixel HS, Siegler AM, Messori L, van Kasteren SI, Bonnet S (2020) Alkyne functionalization of a photoactivated ruthenium polypyridyl complex for click-enabled serum albumin interaction studies. Inorg Chem 59:7710–7720. https://doi.org/10.1021/acs.inorgchem.0c00742

Lomelino CL, Mahon BP, McKenna R, Carta F, Supuran CT (2016) Kinetic and X-ray crystallographic investigations on carbonic anhydrase isoforms I, II, IX and XII of a thioureido analog of SLC-0111. Bioorganic Med Chem 24:976–981. https://doi.org/10.1016/j.bmc.2016.01.019

Nguyen GTH, Tran TN, Podgorski MN, Bell SG, Supuran CT, Donald WA (2019) Nanoscale ion emitters in native mass spectrometry for measuring ligand-protein binding affinities. ACS Cent Sci 5:308–318. https://doi.org/10.1021/acscentsci.8b00787

Nguyen GTH, Nocentini A, Angeli A, Gratteri P, Supuran CT, Donald WA (2020) Perfluoroalkyl substances of significant environmental concern can strongly inhibit human carbonic anhydrase isozymes. Anal Chem 92:4614–4622. https://doi.org/10.1021/acs.analchem.0c00163

De Simone G, Langella E, Esposito D, Supuran CT, Monti SM, Winum J-Y, Alterio V (2017) Insights into the binding mode of sulphamates and sulphamides to hCA II: crystallographic studies and binding free energy calculations. J Enzyme Inhib Med Chem 32:1002–1011. https://doi.org/10.1080/14756366.2017.1349764

Bachmann KA, Lewis JD (2005) Predicting inhibitory drug—drug interactions and evaluating drug interaction reports using inhibition constants. Ann Pharmacother 39:1064–1072. https://doi.org/10.1345/aph.1E508

El-Azab AS, Abdel-Aziz AAM, Bua S, Nocentini A, AlSaif NA, Almehizia AA, Alanazi MM, Hefnawy MM, Supuran CT (2019) New anthranilic acid-incorporating N-benzenesulfonamidophthalimides as potent inhibitors of carbonic anhydrases I, II, IX, and XII: synthesis, in vitro testing, and in silico assessment. Eur J Med Chem 181:111573. https://doi.org/10.1016/j.ejmech.2019.111573

Brzozowski Z, Sławiński J, Vullo D, Supuran CT (2012) Carbonic anhydrase inhibitors. Regioselective synthesis of novel series 1-substituted 1,4-dihydro-4-oxo-3-pyridinesulfonamides and their inhibition of the human cytosolic isozymes I and II and transmembrane cancer-associated isozymes IX and XII. Eur J Med Chem 56:282–291. https://doi.org/10.1016/j.ejmech.2012.08.006

Marzo T, Massai L, Pratesi A, Stefanini M, Cirri D, Magherini F, Becatti M, Landini I, Nobili S, Mini E, Crociani O, Arcangeli A, Pillozzi S, Gamberi T, Messori L (2019) Replacement of the thiosugar of auranofin with iodide enhances the anticancer potency in a mouse model of ovarian cancer. ACS Med Chem Lett 10:656–660. https://doi.org/10.1021/acsmedchemlett.9b00007

Landini I, Lapucci A, Pratesi A, Massai L, Napoli C, Perrone G, Pinzani P, Messori L, Mini E, Nobili S (2017) Selection and characterization of a human ovarian cancer cell line resistant to auranofin. Oncotarget 8:96062–96078. https://doi.org/10.18632/oncotarget.21708

Maurais AJ, Weerapana E (2019) Reactive-cysteine profiling for drug discovery. Curr Opin Chem Biol 50:29–36

Xu K, Zhang Y, Tang B, Laskin J, Roach PJ, Chen H (2010) Study of highly selective and efficient thiol derivatization using selenium reagents by mass spectrometry. Anal Chem 82:6926–6932. https://doi.org/10.1021/ac1011602

Capper MJ, Wright GSA, Barbieri L, Luchinat E, Mercatelli E, McAlary L, Yerbury JJ, O’Neill PM, Antonyuk SV, Banci L, Hasnain SS (2018) The cysteine-reactive small molecule ebselen facilitates effective SOD1 maturation. Nat Commun. https://doi.org/10.1038/s41467-018-04114-x

Terentis AC, Freewan M, Sempértegui Plaza TS, Raftery MJ, Stocker R, Thomas SR (2010) The selenazal drug ebselen potently inhibits indoleamine 2,3-dioxygenase by targeting enzyme cysteine residues. Biochemistry 49:591–600. https://doi.org/10.1021/bi901546e

Müller A, Cadenas E, Graf P, Sies H (1984) A novel biologically active seleno-organic compound-1. Glutathione peroxidase-like activity in vitro and antioxidant capacity of PZ 51 (Ebselen). Biochem Pharmacol 33:3235–3239. https://doi.org/10.1016/0006-2952(84)90083-2

Isab AA, Frank Shaw C, Lockela J, Hoeschele JD (1988) Reactions of trimethylphosphine analogues of auranofin with bovine serum albumin. Inorg Chem 27:3588–3592. https://doi.org/10.1021/ic00293a029

Carlsson U, Aasa R, Jonsson B-H, Lindskog S, Henderson LE (1975) Paramagnetic and fluorescent probes attached to “buried” sulfhydryl groups in human carbonic anhydrases. Application to inhibitor binding, denaturation and refolding. Eur J Biochem 52:25–36. https://doi.org/10.1111/j.1432-1033.1975.tb03969.x

Rana TM, Meares CF (1991) Transfer of oxygen from an artificial protease to peptide carbon during proteolysis. Proc Natl Acad Sci USA 88:10578–10582