- Email: [email protected]
The conversion of azo-quenchers to fluorophores
Analytical Biochemistry 585 (2019) 113400
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
The conversion of azo-quenchers to fluorophores a
b
c,∗
T
c
O. Hofstetter , H. Hofstetter , T. Miron , M. Wilchek a b c
Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL, 60115, USA Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA Department of Biomolecular Science, Weizmann institute of Science, Rehovot, Israel
A R T I C LE I N FO
A B S T R A C T
Keywords: Quencher Fluorophore FRET Beacon Azo dye
In this short note we describe the conversion of the widely used fluorescence quenching azo-dyes DABCYL and HABA to fluorophores. The dyes were conjugated to the proteins RNase and human serum albumin (HSA) and subsequently reduced using sodium dithionite (Na2S2O4), thus forming amine-containing fluorophores. Since this chemical reaction can be applied to any azo-containing quencher compound, a great variety of substances can be readily obtained synthetically. This approach provides a promising tool in the use of fluorescence-based investigations of biomolecular interactions.
1. Introduction Fluorescence-based methods have become indispensable in chemistry, physics, biology and biotechnology, and are essential to studying biological processes both in vitro and in vivo [1–3]. Fluorescent dyes can be readily coupled to biologically active molecules such as proteins, nucleic acids and polysaccharides, and even whole cells [3,4]. The simplest and earliest applications of fluorescent probes utilized tryptophan and tyrosine residues of peptides [5] and proteins, or covalently bound fluorophores on proteins, attached to the amino acid side chains of lysines, cysteines, and glutamic acids, in order to study their proximal environment [6]. Thus, local structure dynamics and conformational changes in proteins could be determined [2]. More recently, protein folding and protein-protein interactions have been investigated by introducing more than one fluorophore or quencher [7,8]. Widely used methodologies include Förster Resonance Energy Transfer (FRET) [5,9], utilizing two fluorophores, and molecular beacons [10,11], containing a fluorophore and a quencher. Applying these methods enables the investigation of conformational changes of biomolecules, as well as measuring the distances and interactions between proteins, nucleic acids, and the hybridization of DNA-strands [11,12]. Both methods are based on energy transfer between two labels that are in close proximity [13]. As demonstrated by some of us earlier, the effect rapidly decreases as the distance between the fluorophores and quenchers increases [5]. In the case of molecular beacons, the quencher is typically a dye
that does not emit fluorescence by itself, but serves as a sink of energy, as is the case with the most commonly used quencher, the azo-dye 4-[4(Dimethylamino)phenylazo]-benzoic acid [DABCYL]. We realized that the conversion of quenchers to non-quenchers, or even to fluorophores, might greatly broaden the range of fluorescencebased labels and their applications, and possibly diminish problems of non-specific quenching of other chromophores present within the molecules under investigation. To demonstrate the feasibility of this approach, we first coupled the azo-dye quenchers DABCYL and 2-(4-hydroxyphenylazo)benzoic acid (HABA) to proteins, and subsequently reduced the conjugates with sodium dithionite to yield 4-aminobenzoyl-and 2-aminobenzoyl-derivatized fluorescent proteins, respectively (Scheme 1). The reaction shown in Scheme 1 is based on our earlier studies [14], in which 3-azotyrosines in proteins were converted to 3-aminotyrosines by reduction with sodium dithionite. Ribonuclease and human serum albumin (HSA), at concentrations of 2–20 mg/mL in PBS, pH 7.4, were reacted with DABCYL-N-hydroxysuccinimide ester (DABCYL-NHS; Merck, Darmstadt, Germany) at a molar ratio between 4 and 50/protein according to the instructions of the supplier. The reactions were carried out at 4 °C overnight. After attaching the azo-label, the protein-derivatives were purified from excess azo-dye by size exclusion chromatography using Sephadex G-25. At this stage, both proteins were colored and displayed maximum light absorption at around 390 nm (Scheme 1A, Fig. 1 insert). However, no fluorescence was observed (Fig. 1a). The proteins were then reduced in
Abbreviations: DABCYL, 4-[4-(Dimethylamino)phenylazobenzoic acid; HABA, 2-(4-hydroxyphenylazo)benzoic acid; HSA, human serum albumin; FRET, Förster Resonance Energy Transfer; Abz, 2-amino benzoyl; NHS, N-hydroxysuccinimide ⁎ Corresponding author. E-mail address: [email protected] (T. Miron). https://doi.org/10.1016/j.ab.2019.113400 Received 15 July 2019; Received in revised form 19 August 2019; Accepted 19 August 2019 Available online 19 August 2019 0003-2697/ © 2019 Elsevier Inc. All rights reserved.
Analytical Biochemistry 585 (2019) 113400
O. Hofstetter, et al.
Scheme 1. Reduction of azo-containing proteins with Na2S2O4. A: Reduction of DABCYL-proteins; B: Reduction of HABA-proteins; P-protein.
Fig. 2. Fluorescence of RNase (a) HABA- RNase (b), Na2S2O4-reduced HABARNase (c). Excitation wavelength was 315 nm [16].
Fig. 1. Fluorescence spectra of DABCYL-RNase (a), Na2S2O4-reduced DABCYLRNase (b). Insert: Absorption spectrum in PBS (A) of DABCYL-RNase (B). In all fluorescence experiments, the absorbance of the samples at 290 nm was in the range of 0.05–0.1 OD. The excitation wavelength was set to 275 nm. Emission scans were recorded between 300 and 600 nm.
2
Analytical Biochemistry 585 (2019) 113400
O. Hofstetter, et al.
a slightly decreased intensity, and a new fluorescence emission peak of Abz appeared around 410 nm (Fig. 3c). The decrease observed in Trp fluorescence may be due to energy transfer from the Trp to the Abz modified HSA. There is a spectral overlap integral between the donor fluorescence spectrum of Trp and the absorption spectrum of Abz at 315 nm (Scheme 1B). The Abz now become an acceptor. Since it is possible to prepare an almost unlimited number of azocontaining reagents with different functional groups and optical properties [13,17], and convert them into amino containing aromatic derivatives after dithionite reduction, a great variety of protein-labels that can be utilized to modulate fluorescent properties of biomolecules can be generated. In conclusion, this short study opens up new approaches and tools to investigate protein folding and biomolecular interactions, and expands existing spectroscopic techniques and probes such as FRET and molecular beacons. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ab.2019.113400. References [1] E.A. Jares-Erijman, T.M. Jovin, FRET imaging, Nat. Biotechnol. 21 (2003) 1387–1395. [2] E. Haas, Ensemble FRET methods in studies of intrinsically disordered proteins, Methods Mol. Biol. 895 (2000) 467–498. [3] M. Whitaker, Fluorescent tags of protein function in living cells, Bioessays 22 (2000) 180–187. [4] B.T. Bajar, E.S. Wang, S. Zhang, M.Z. Lin, J. Chu, A guide to fluorescent protein FRET pairs, Sensors 16 (2016) 1488, https://doi.org/10.3390/s16091488 PMCID: PMC5038762 PMID: 27649177 Published online 2016 Sep. 14. [5] H. Edelhoch, L. Brand, M. Wilchek, Fluorescence studies with tryptophyl peptides, Biochemistry 6 (1967) 547–559. [6] H. Haas, M. Wilchek, E. Katchalski-Katzir, I.Z. Steinberg, Distribution of end-to-end distances of oligopeptides in solution as estimated by energy transfer, Proc. Natl. Acad. Sci. 72 (1975) 1807–1811. [7] B. Wallace, P.J. Atzberger, Förster resonance energy transfer: role of diffusion of fluorophore orientation and separation in observed shifts of FRET efficiency, PLoS One 12 (2017) e0177122https://doi:10.1371/journal.pone. 0177122PMCID:PMC5438121. [8] D.W. Piston, G.J. Kremers, Fluorescent protein FRET: the good, the bad and the ugly, Trends Biochem. Sci. 32 (2007) 407–414. [9] R.M. Clegg, FRET tells us about proximities, distances, orientations and dynamic properties, J. Biotechnol. 82 (2002) 177–179. [10] S. Tyagi, F.R. Kramer, Molecular beacons: probes that fluoresce upon hybridization, Nat. Biotechnol. 14 (1996) 303–308. [11] S.A.E. Marras, Selection of fluorophore and quencher pairs for fluorescent nucleic acid hybridization probes, Methods Mol. Biol. 335 (2006) 3–16. [12] F. Fäßler, P. Pimpl, In vivo interaction studies by measuring Förster resonance energy transfer through fluorescence lifetime imaging microscopy (FRET/FLIM), Methods Mol. Biol. 1662 (2017) 159–170. [13] P. Crisalli, E.T. Kool, Multi-path quenchers: efficient quenching of common fluorophores, Bioconjug. Chem. 22 (2011) 2345–2354. [14] M. Gorecki, M. Wilchek, A. Patchornik, The conversion of 3-monoazotyrosine to 3aminotyrosine in peptides and proteins, Biochim. Biophys. Acta 229 (1971) 590–595. [15] N. Nishino, Y. Makinose, T. Fugimoto, 2-aminobenzoyl-peptide-2,4-dinitroanilinoethylamides, Facile fluorescent detection system for sequence specific proteases, Chem. Lett. 77 (1992) 77–80. [16] A.S. Ito, R. De F. Turchiello, I.Y. Hirata, M.H.S. Cezari, M. Meldal, L. Juliano, Fluorescent properties of amino acids labeled with ortho-aminobenzoic acid, Biospectroscopy 4 (1998) 395–402. [17] R.M. Christie, Azo dyes and pigments (chapter 3), in: R.M. Christie (Ed.), Colour Chemistry, Royal society of Chemistry, UK, 2001, pp. 45–68.
Fig. 3. Fluorescence of: HSA (a) HABA-HSA (b) Na2S2O4-reduced HABA-HSA (c). Excitation wavelength at 315 nm [16].
PBS or water by adding sodium dithionite (1 mg/mg protein) for 30 min, upon which the color of the conjugates completely disappeared. Excess of the reducing agent and the cleavage product was removed from the reduced, labeled proteins, by size exclusion chromatography. The absorbance maximum of the resulting modified proteins was between 270 and 290 nm as indicated in Scheme 1. Furthermore, these proteins were highly fluorescent with an emission maximum at 357 nm (Fig. 1b), as was demonstrated for Ribonuclease (RNase). RNase was chosen for this particular experiment because it has no tryptophan, which emits fluorescent light in this range (λmax 340–350 nm). In order to shift the fluorescence of the reduced protein-conjugates to a wavelength higher than that of protein autofluorescence and to develop a known fluorophore, such as 2-aminobenzoyl (Abz) [15,16], which absorbs around 315 nm and fluorescence at around 410 nm. We coupled HABA to HSA and Ribonuclease following the protocol described for DABCYL-NHS. The HABA-NHS used was prepared by coupling HABA with N-hydroxysuccinimide in DMF in the presence of N,N′-Dicyclohexylcarbodiimide (DCC) at equimolar amounts. Dithionite reduction of the HABA-derivatized proteins yielded Abz proteins (Scheme 1B). Modification of Ribonuclease with HABA resulted in a colored protein which absorbed light at 350 nm and was not fluorescent (Fig. 2 a). Upon reduction, the Abz-derivatized RNase became highly fluorescent with an emission maximum around 410 nm and absorption spectrum around 315 nm as shown in Ref. [16]. This reaction allows the introduction of fluorophores into non-fluorescent molecules. When HABA was coupled to HSA, the auto-fluorescence of the Trp was quenched due to overlapping spectra of HABA absorption (Fig. 3b) at 350 nm and Trp fluorescence at the same wavelength (Fig. 3a). Reduction with dithionite, however, recovered the Trp fluorescence, with
3
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
The conversion of azo-quenchers to fluorophores a
b
c,∗
T
c
O. Hofstetter , H. Hofstetter , T. Miron , M. Wilchek a b c
Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL, 60115, USA Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA Department of Biomolecular Science, Weizmann institute of Science, Rehovot, Israel
A R T I C LE I N FO
A B S T R A C T
Keywords: Quencher Fluorophore FRET Beacon Azo dye
In this short note we describe the conversion of the widely used fluorescence quenching azo-dyes DABCYL and HABA to fluorophores. The dyes were conjugated to the proteins RNase and human serum albumin (HSA) and subsequently reduced using sodium dithionite (Na2S2O4), thus forming amine-containing fluorophores. Since this chemical reaction can be applied to any azo-containing quencher compound, a great variety of substances can be readily obtained synthetically. This approach provides a promising tool in the use of fluorescence-based investigations of biomolecular interactions.
1. Introduction Fluorescence-based methods have become indispensable in chemistry, physics, biology and biotechnology, and are essential to studying biological processes both in vitro and in vivo [1–3]. Fluorescent dyes can be readily coupled to biologically active molecules such as proteins, nucleic acids and polysaccharides, and even whole cells [3,4]. The simplest and earliest applications of fluorescent probes utilized tryptophan and tyrosine residues of peptides [5] and proteins, or covalently bound fluorophores on proteins, attached to the amino acid side chains of lysines, cysteines, and glutamic acids, in order to study their proximal environment [6]. Thus, local structure dynamics and conformational changes in proteins could be determined [2]. More recently, protein folding and protein-protein interactions have been investigated by introducing more than one fluorophore or quencher [7,8]. Widely used methodologies include Förster Resonance Energy Transfer (FRET) [5,9], utilizing two fluorophores, and molecular beacons [10,11], containing a fluorophore and a quencher. Applying these methods enables the investigation of conformational changes of biomolecules, as well as measuring the distances and interactions between proteins, nucleic acids, and the hybridization of DNA-strands [11,12]. Both methods are based on energy transfer between two labels that are in close proximity [13]. As demonstrated by some of us earlier, the effect rapidly decreases as the distance between the fluorophores and quenchers increases [5]. In the case of molecular beacons, the quencher is typically a dye
that does not emit fluorescence by itself, but serves as a sink of energy, as is the case with the most commonly used quencher, the azo-dye 4-[4(Dimethylamino)phenylazo]-benzoic acid [DABCYL]. We realized that the conversion of quenchers to non-quenchers, or even to fluorophores, might greatly broaden the range of fluorescencebased labels and their applications, and possibly diminish problems of non-specific quenching of other chromophores present within the molecules under investigation. To demonstrate the feasibility of this approach, we first coupled the azo-dye quenchers DABCYL and 2-(4-hydroxyphenylazo)benzoic acid (HABA) to proteins, and subsequently reduced the conjugates with sodium dithionite to yield 4-aminobenzoyl-and 2-aminobenzoyl-derivatized fluorescent proteins, respectively (Scheme 1). The reaction shown in Scheme 1 is based on our earlier studies [14], in which 3-azotyrosines in proteins were converted to 3-aminotyrosines by reduction with sodium dithionite. Ribonuclease and human serum albumin (HSA), at concentrations of 2–20 mg/mL in PBS, pH 7.4, were reacted with DABCYL-N-hydroxysuccinimide ester (DABCYL-NHS; Merck, Darmstadt, Germany) at a molar ratio between 4 and 50/protein according to the instructions of the supplier. The reactions were carried out at 4 °C overnight. After attaching the azo-label, the protein-derivatives were purified from excess azo-dye by size exclusion chromatography using Sephadex G-25. At this stage, both proteins were colored and displayed maximum light absorption at around 390 nm (Scheme 1A, Fig. 1 insert). However, no fluorescence was observed (Fig. 1a). The proteins were then reduced in
Abbreviations: DABCYL, 4-[4-(Dimethylamino)phenylazobenzoic acid; HABA, 2-(4-hydroxyphenylazo)benzoic acid; HSA, human serum albumin; FRET, Förster Resonance Energy Transfer; Abz, 2-amino benzoyl; NHS, N-hydroxysuccinimide ⁎ Corresponding author. E-mail address: [email protected] (T. Miron). https://doi.org/10.1016/j.ab.2019.113400 Received 15 July 2019; Received in revised form 19 August 2019; Accepted 19 August 2019 Available online 19 August 2019 0003-2697/ © 2019 Elsevier Inc. All rights reserved.
Analytical Biochemistry 585 (2019) 113400
O. Hofstetter, et al.
Scheme 1. Reduction of azo-containing proteins with Na2S2O4. A: Reduction of DABCYL-proteins; B: Reduction of HABA-proteins; P-protein.
Fig. 2. Fluorescence of RNase (a) HABA- RNase (b), Na2S2O4-reduced HABARNase (c). Excitation wavelength was 315 nm [16].
Fig. 1. Fluorescence spectra of DABCYL-RNase (a), Na2S2O4-reduced DABCYLRNase (b). Insert: Absorption spectrum in PBS (A) of DABCYL-RNase (B). In all fluorescence experiments, the absorbance of the samples at 290 nm was in the range of 0.05–0.1 OD. The excitation wavelength was set to 275 nm. Emission scans were recorded between 300 and 600 nm.
2
Analytical Biochemistry 585 (2019) 113400
O. Hofstetter, et al.
a slightly decreased intensity, and a new fluorescence emission peak of Abz appeared around 410 nm (Fig. 3c). The decrease observed in Trp fluorescence may be due to energy transfer from the Trp to the Abz modified HSA. There is a spectral overlap integral between the donor fluorescence spectrum of Trp and the absorption spectrum of Abz at 315 nm (Scheme 1B). The Abz now become an acceptor. Since it is possible to prepare an almost unlimited number of azocontaining reagents with different functional groups and optical properties [13,17], and convert them into amino containing aromatic derivatives after dithionite reduction, a great variety of protein-labels that can be utilized to modulate fluorescent properties of biomolecules can be generated. In conclusion, this short study opens up new approaches and tools to investigate protein folding and biomolecular interactions, and expands existing spectroscopic techniques and probes such as FRET and molecular beacons. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ab.2019.113400. References [1] E.A. Jares-Erijman, T.M. Jovin, FRET imaging, Nat. Biotechnol. 21 (2003) 1387–1395. [2] E. Haas, Ensemble FRET methods in studies of intrinsically disordered proteins, Methods Mol. Biol. 895 (2000) 467–498. [3] M. Whitaker, Fluorescent tags of protein function in living cells, Bioessays 22 (2000) 180–187. [4] B.T. Bajar, E.S. Wang, S. Zhang, M.Z. Lin, J. Chu, A guide to fluorescent protein FRET pairs, Sensors 16 (2016) 1488, https://doi.org/10.3390/s16091488 PMCID: PMC5038762 PMID: 27649177 Published online 2016 Sep. 14. [5] H. Edelhoch, L. Brand, M. Wilchek, Fluorescence studies with tryptophyl peptides, Biochemistry 6 (1967) 547–559. [6] H. Haas, M. Wilchek, E. Katchalski-Katzir, I.Z. Steinberg, Distribution of end-to-end distances of oligopeptides in solution as estimated by energy transfer, Proc. Natl. Acad. Sci. 72 (1975) 1807–1811. [7] B. Wallace, P.J. Atzberger, Förster resonance energy transfer: role of diffusion of fluorophore orientation and separation in observed shifts of FRET efficiency, PLoS One 12 (2017) e0177122https://doi:10.1371/journal.pone. 0177122PMCID:PMC5438121. [8] D.W. Piston, G.J. Kremers, Fluorescent protein FRET: the good, the bad and the ugly, Trends Biochem. Sci. 32 (2007) 407–414. [9] R.M. Clegg, FRET tells us about proximities, distances, orientations and dynamic properties, J. Biotechnol. 82 (2002) 177–179. [10] S. Tyagi, F.R. Kramer, Molecular beacons: probes that fluoresce upon hybridization, Nat. Biotechnol. 14 (1996) 303–308. [11] S.A.E. Marras, Selection of fluorophore and quencher pairs for fluorescent nucleic acid hybridization probes, Methods Mol. Biol. 335 (2006) 3–16. [12] F. Fäßler, P. Pimpl, In vivo interaction studies by measuring Förster resonance energy transfer through fluorescence lifetime imaging microscopy (FRET/FLIM), Methods Mol. Biol. 1662 (2017) 159–170. [13] P. Crisalli, E.T. Kool, Multi-path quenchers: efficient quenching of common fluorophores, Bioconjug. Chem. 22 (2011) 2345–2354. [14] M. Gorecki, M. Wilchek, A. Patchornik, The conversion of 3-monoazotyrosine to 3aminotyrosine in peptides and proteins, Biochim. Biophys. Acta 229 (1971) 590–595. [15] N. Nishino, Y. Makinose, T. Fugimoto, 2-aminobenzoyl-peptide-2,4-dinitroanilinoethylamides, Facile fluorescent detection system for sequence specific proteases, Chem. Lett. 77 (1992) 77–80. [16] A.S. Ito, R. De F. Turchiello, I.Y. Hirata, M.H.S. Cezari, M. Meldal, L. Juliano, Fluorescent properties of amino acids labeled with ortho-aminobenzoic acid, Biospectroscopy 4 (1998) 395–402. [17] R.M. Christie, Azo dyes and pigments (chapter 3), in: R.M. Christie (Ed.), Colour Chemistry, Royal society of Chemistry, UK, 2001, pp. 45–68.
Fig. 3. Fluorescence of: HSA (a) HABA-HSA (b) Na2S2O4-reduced HABA-HSA (c). Excitation wavelength at 315 nm [16].
PBS or water by adding sodium dithionite (1 mg/mg protein) for 30 min, upon which the color of the conjugates completely disappeared. Excess of the reducing agent and the cleavage product was removed from the reduced, labeled proteins, by size exclusion chromatography. The absorbance maximum of the resulting modified proteins was between 270 and 290 nm as indicated in Scheme 1. Furthermore, these proteins were highly fluorescent with an emission maximum at 357 nm (Fig. 1b), as was demonstrated for Ribonuclease (RNase). RNase was chosen for this particular experiment because it has no tryptophan, which emits fluorescent light in this range (λmax 340–350 nm). In order to shift the fluorescence of the reduced protein-conjugates to a wavelength higher than that of protein autofluorescence and to develop a known fluorophore, such as 2-aminobenzoyl (Abz) [15,16], which absorbs around 315 nm and fluorescence at around 410 nm. We coupled HABA to HSA and Ribonuclease following the protocol described for DABCYL-NHS. The HABA-NHS used was prepared by coupling HABA with N-hydroxysuccinimide in DMF in the presence of N,N′-Dicyclohexylcarbodiimide (DCC) at equimolar amounts. Dithionite reduction of the HABA-derivatized proteins yielded Abz proteins (Scheme 1B). Modification of Ribonuclease with HABA resulted in a colored protein which absorbed light at 350 nm and was not fluorescent (Fig. 2 a). Upon reduction, the Abz-derivatized RNase became highly fluorescent with an emission maximum around 410 nm and absorption spectrum around 315 nm as shown in Ref. [16]. This reaction allows the introduction of fluorophores into non-fluorescent molecules. When HABA was coupled to HSA, the auto-fluorescence of the Trp was quenched due to overlapping spectra of HABA absorption (Fig. 3b) at 350 nm and Trp fluorescence at the same wavelength (Fig. 3a). Reduction with dithionite, however, recovered the Trp fluorescence, with
3