Quenching of tryptophan fluorescence in various proteins by a series of small nickel complexes

Article abstract of DOI:10.1039/c2dt12169g

A series of twelve anionic, cationic, and neutral nickel(ii) complexes have been synthesized and characterized. The interaction of these complexes with bovine serum albumin (BSA), human serum albumin (HSA), lysozyme (Lyso), and tryptophan (Trp) has been studied using steady-state fluorescence spectroscopy. Dynamic and static quenching constants have been calculated, and the role played in quenching by the ligand and complex charge investigated. The nickel complexes showed selectivity towards the different proteins based on the environment surrounding the Trp residue(s). Only small neutral complexes with hydrophobic ligands effectively quenched protein fluorescence via static quenching, with association constants ranging from 10² M^-1 (free Trp) to 10¹⁰ M^-1 (lysozyme), indicating a spontaneous and thermodynamically favorable interaction. The number of binding sites, on average, was determined to be one in BSA, HSA and free Trp, and two in lysozyme. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2dt12169g

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PAPER
Quenching of tryptophan fluorescence in various proteins by a series of small
nickel complexes†
Heather F. Crouse,a Julianne Potoma,a Farhat Nejrabi,b Deanna L. Snyder,a Balwant S. Chohanb and
Swarna Basu*a
Received 13th November 2011, Accepted 22nd November 2011
DOI: 10.1039/c2dt12169g
A series of twelve anionic, cationic, and neutral nickel(II) complexes have been synthesized and
characterized. The interaction of these complexes with bovine serum albumin (BSA), human serum
albumin (HSA), lysozyme (Lyso), and tryptophan (Trp) has been studied using steady-state fluorescence
spectroscopy. Dynamic and static quenching constants have been calculated, and the role played in
quenching by the ligand and complex charge investigated. The nickel complexes showed selectivity
towards the different proteins based on the environment surrounding the Trp residue(s). Only small
neutral complexes with hydrophobic ligands effectively quenched protein fluorescence via static
quenching, with association constants ranging from 102 M−1 (free Trp) to 1010 M−1 (lysozyme),
indicating a spontaneous and thermodynamically favorable interaction. The number of binding sites, on
average, was determined to be one in BSA, HSA and free Trp, and two in lysozyme.
proteins in the circulatory system.10–13 HSA consists of a single
Introduction
polypeptide chain of 585 amino acids, a scarcity of Trp and Met
residues, and an abundance of Cys and charged residues.14–17
Protein interactions play a fundamental role in many biochemical
processes in both the healthy and the diseased state, including
critical roles in signal transduction, immune reaction, cell cycle
control, differentiation, and protein folding. In order to under-
stand, probe and manipulate biological systems effectively there
is a need to develop small molecules that influence or inhibit
protein interactions through selective recognition.1,2 Small metal
complexes can potentially combine flexibility in ligand design
with access to a wide and diverse range of coordination geome-
tries, optical isomers and electronic states. This flexibility can be
used to enable or prevent coordination of the metal complex to
protein side chains or substrates.
BSA is a homologous globular protein, consisting of 583 amino
acid residues in the mature form, and the sequence of BSA is
76% identical to that of HSA. While the crystal structure of HSA
as well as its binding with fatty acids has been characterized, the
crystallographic structure of BSA has not been determined to
any sufficient resolution.16–19 The albumins are composed of
three structurally similar domains (I, II and III), each containing
two subdomains (A and B) and stabilized by 17 disulphide
bridges.11–15,20–24 The flexible multidomain structures house a
variety of binding sites, and allow for cooperativity and allosteric
modulation, thus making HSA and BSA similar to multimeric
proteins.
Albumins can reversibly bind with a number of molecules in
the plasma through various interactions. Of the two major struc-
turally selective binding sites, the binding affinity offered by site
I is largely through hydrophobic interactions, whereas site II
involves a combination of hydrophobic, hydrogen bonding, and
electrostatic interactions.25,26 Most strongly bound are medium-
sized hydrophobic organic anions, including long-chain fatty
acids, bilirubin and haematin. Hydrated monovalent cations do
not associate to any significant extent, whereas hydrated divalent
cations such as Ca2+ strongly bind at select groups on the
albumin molecule.27 Information about the interaction of ions
surrounded by various types of organic ligands with serum
albumin can help reveal the role of albumins in the normal state
and in the diseased state, and thus provide the basis for the
rational design of efficient drugs, sensitizers, and new thera-
peutic approaches.
A. Serum proteins
Several different transport proteins exist in blood plasma, but
only albumin is able to bind and transport a wide diversity3–6 of
ligands with reversibility and high affinity. For many drugs,
binding to serum albumin is a critical determinant of their distri-
bution and pharmacokinetics.7–9 HSA accounts for 60% of the
measured serum proteins, thus is one of the most abundant
aDepartment of Chemistry, Susquehanna University, 514 University
Avenue Selinsgrove, PA 17870, USA. E-mail: basu@susqu.edu;
Fax: 1-570-372-2752; Tel: 1-570-372-4223
bPenn State Harrisburg, 777 West Harrisburg Pike, Middletown, PA
17057, USA
†Electronic supplementary information (ESI) available. CCDC reference
numbers 830386–830389 and 853856. For ESI and crystallographic data
in CIF or other electronic format see DOI: 10.1039/c2dt12169g
2720 | Dalton Trans., 2012, 41, 2720–2731
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B. Nickel and metal binding
The interaction of metal ions has led to our interest in studying
the interaction of transition metal complexes with proteins. The
primary focus has been on the effect that different ligand
systems have on the interaction, particularly the drug-binding
sites of BSA. Secondly, the possibility that fluorescence quench-
ing of the protein is due to FRET (fluorescence resonance
energy transfer) from the protein to the complex will lead to the
calculation of intermolecular distances to help determine the
proximity of the complex to the albumin proteins.13 A recent
study concerning the interaction of a fluorone-Mo(VI) complex
with HSA and BSA72 showed there were strong and significant
interactions between the metal complex and the protein.73 Others
have recently reported that a Cr(III) complex, ([Cr(salprn)(H2O)2]
ClO4), is capable of quenching BSA via FRET leading to a non-
specific photocleavage of the protein.74,75 We have recently
shown that a Cr(III) complex, (R,R)-N,N′-bis(3,5-di-tert-butyl-
salicylidene)-1,2-cyclohexane-diamino chromium(III), binds very
tightly to serum albumins and lysozyme, and that the binding is
highly specific and the quenching is static in nature and is not
due to energy transfer.76 Lysozyme is included in our studies
since it is well characterized,77–79 and contains multiple Trp resi-
dues that are important for enzymatic activity.80 It is a single
polypeptide chain of 129 amino acid residues, six of which are
Trp residues at positions 28, 62, 63, 108, 111, and 123. The resi-
dues at 62, 63, and 108 are located deep in the active site. It is
expected that each of the residues will be affected differently
depending upon their accessibility, and thus contributing inde-
pendently to the inherent fluorescence intensity.71,77,80–83
Metals have important roles in biological systems ranging from
essential to toxic, and as such the metal-binding capacity of
albumins has been widely studied over the years,28–33 including
the affinity, stoichiometry and specificity of Ca(II), Co(II), Ni(II),
Cu(II), Zn(II), Cd(II), Cr(III), V(III), and Mo(VI) ions.34–46
Although there is a lack of detailed structural information, four
different metal sites have been described, and specificity is
achieved by exploiting differences in the co-ordination chem-
istries of the preferred metal ions: (i) ATCUN site (N-terminal
Cu/Ni-binding) motif at the N-terminus, the primary site47 for
Cu(II) and Ni(II); (ii) site A, the primary Zn(II)-binding site6 that
can also bind other +2 metal ions; (iii) site B, which displays a
high affinity towards Cd(II), the location of which is
unknown;36,38,40 and (iv) the reduced thiol of Cys-34, which
binds gold48 and platinum49,50 compounds.
Nickel enzymes were first discovered51 in the 1970’s, and
although studies in rats indicate that Ni is an essential element
for higher animals,52,53 neither the source of the Ni requirement
nor a single mammalian Ni-dependent enzyme has been ident-
ified. Although pure Ni itself is not toxic, Ni complexes can be
highly toxic especially with long term contact exposure in
humans.54,55 Allergy to Ni is a phenomenon which has assumed
growing importance in recent years.56 According to current
models, haptens penetrating the epidermis bind to carrier pro-
teins found just under the skin, leading to an inflammatory reac-
tion within the skin.57–63 The role played by albumins in
translocating Ni2+ toward deeper epidermal layers has not been
satisfactorily resolved, though it is of interest that (1) HSA is
particularly abundant in skin, (2) it is a transporter of essential
nutrients for epithelial cells in the absence of blood vessels, and
(3) it is able to efficiently cross the epidermal basement mem-
brane.64 The investigation of the interaction of Ni complexes
with serum albumins is imperative.
In this work, we have studied the interaction between a series
of Ni(II) complexes (Fig. 1) and lysozyme, HSA and BSA, using
fluorescence and UV-vis spectroscopic techniques under physio-
logical conditions. The interaction between the Ni(II) complexes
and free Trp was also investigated to illustrate the affinity of the
complex for this particular amino acid residue, irrespective of
environment. The results have allowed us to discuss the type,
degree and nature of the interaction and binding between the
protein and metal complexes with consideration to polarity,
hydrophobicity, charge, and spin state of the metal complex.
C. Fluorimetry
One way to measure the extent and accessibility of protein
binding sites to small molecules involves fluorescence quench-
ing. Information concerning the number of binding sites, the
binding constant Ka, and the distance between donor and accep-
tor can be readily obtained using Stern–Volmer kinetics and
Forster’s theory.65–68 The fluorescence signature of a protein
derives from the aromatic residues. The fluorescence from the
indole group in Trp is extremely sensitive to its environment,
and is a convenient spectroscopic probe for the structure and
rotational dynamics surrounding the Trp residue.66,69 These Trp
residues can undergo electronic excitation in the visible region,
and the emission can be quenched via different mechanisms
depending on the degree of “exposure” to the quencher.70,71
Therefore, changes in the intrinsic fluorescence and the bi-
molecular quenching rate constant can be used to monitor struc-
tural changes in a protein, and reveal information on Trp
accessibility and how deep it is buried in the protein.66 In HSA,
a Trp residue at position 214 is buried in the second helix of
the second domain, in a hydrophobic fold. BSA has an
additional Trp in position 131 which is found on the surface of
the protein.13
Experimental
A. General procedures
Unless otherwise mentioned, all chemicals were of analytical
reagent grade (Sigma-Aldrich or VWR). Synthetic procedures
and manipulations were conducted using Schlenk techniques,
under an Ar atmosphere. The solvents were either HPLC-grade
or were dried and distilled using conventional84,85 methods and
stored under an Ar atmosphere. All proteins and Trp were pur-
chased from Sigma-Aldrich and used without further purifi-
cation. Stock solutions of proteins, Trp and Ni(II) complexes
were prepared in 50 mM Tris-HCl buffer solution (pH 7.4). Syn-
thesis and characterization data for all complexes (except for per-
tinent X-ray crystallography data) is reported in the SI (#1).†
B. Physical measurements: characterization
NMR spectra were recorded at room temperature on a Anasazi-
Varian 60 MHz FT spectrometer at Penn State Harrisburg, or a
This journal is © The Royal Society of Chemistry 2012
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Fig. 1 Ni(II) complexes used in this study, 1. Ni-Pr-Et. 2. Ni-Pr-Pr. 3. Ni-Pr-Pr-Iodo. 4. Ni(pya2tn)Cl2·H2O. 5. Ni-(en)2Cl2. 6. Ni-Pr-Et-Iodo. 7. Ni
(pya2tn-OH)Cl2. 8. Ni(EDTA)-H2O-Na. 9. Ni-Et-Et. 10. Ni-Et-Pr. 11. Ni(en)3Cl2·2H2O. 12. Ni(NH3)6Cl2. [Pr = propyl, Et = ethyl, en = ethylenedi-
amine, tn = trimethylene diamine, EDTA = ethylenediaminetetraacetate, pya = Schiff base ligand derived from pyridine-2-carboxaldehyde and 1,3-
diaminopropane (pya2tn)].
Bruker AC200 FT at UMass-Amherst, or a Bruker 500 MHz FT
spectrometer at Penn State Hershey Medical Center. Chemical
shifts are reported in ppm relative to an internal standard of
TMS. FTIR data for solid samples (as KBr disks) and liquid
samples (neat) were obtained on a Jasco 4000 or a Perkin Elmer
783 IR spectrometer. The IR spectral peaks were calibrated with
polystyrene, and are reported in wavenumbers. Relative intensi-
ties of the bands in a spectrum are indicated (w = weak, m =
medium, s = strong, vs = very strong, br = broad). Solid-state
magnetic susceptibilities were obtained on Johnson Matthey
(MK-1 and MSB-1) magnetic susceptibility Gouy balances. The
Evans technique for measurement of solution magnetic moments
was also employed,86,87 using coaxial NMR tubes. Electrospray
mass spectrometry (ESI-MS and (MS)n) measurements were
made at UMass using an Esquire LC/MS 32 instrument, or at
Penn State using an Applied Biosystems API 150EX instrument.
The m/z values are given to 4 significant figures. The reported
m/z values in the synthetic section correspond to the most intense
peak in the isotopic cluster, and the calculated and theoretical
m/z values are based on isotopic masses (on the weight of the
most common isotopes, monoisotopic mass). In some cases the
‘most intense peak’ value and the calculated ‘common isotope’
value may not necessarily be the same. Samples for ESI-MS
analysis were introduced as MeOH/MeCN solutions, and
routinely characterized in a combination of both positive and
negative modes. Isotopic MS clusters were identified, and all
those highlighted were found to be in accordance with their
simulations. Selected simulated spectra and the regions that they
represent in the original spectra are included in the SI.† Spectra
were simulated using various MS calculating software programs.
Appropriate coupled MS/MS and (MS)n methods were utilized
where necessary, and both the parent spectra and the fragmenta-
tion patterns were used to elucidate the reaction products. The
experimental data indicate the successful preparation of the com-
plexes mentioned. Elemental analyses were performed at the
UMass-Amherst Microanalysis Center, and QTI, Whitehouse,
NJ. Samples for microanalysis were ground to a fine powder
and vacuum desiccated over phosphorus pentoxide prior to
analysis.
Single-crystal X-ray crystallographic studies were performed
in part by Dr Carl Carrano and Mr J. Hoffman at San Diego
State University using a Bruker X8 Apex diffractometer
(Complex 2), by Dr H. Yennawar using a Bruker Apex SMART
CCD diffractometer at Penn State University (Complexes 3, 8
and 10), and by Dr A. Chandrasekaran at UMass-Amherst using
a Nonius Kappa CCD diffractometer (Complex 6). For each
apparatus, a graphite monochromated Mo-Kα radiation (λ =
0.71073 Å) source was employed. Data were generally collected
at 23 2 °C (specific details are given in the SI†). A θ–2θ scan
mode was used for data collection from about 2° ≤ 2θMo-Kα
≤
29°. The data were solved by a full matrix least squares refine-
ment on F2 using the SHELX suite of programs.88 The plotting
2722 | Dalton Trans., 2012, 41, 2720–2731
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Table 1 A comparison of selected bond lengths (Å) and bond angles (°) for complexes 1,97 6, 2, 3, 9,98 and 10
Bond lengths (Å)
1
6
2
3
9
10a
Ni–S1
Ni–S2
Ni–N1
Ni–N2
Ni–O1
Ni–O2
2.176(1)
2.174(1)
1.999(3)
2.006(3)
2.407(4)
2.446(3)
2.098(10)
2.118(10)
2.134(8)
2.035(8)
2.1605(3)
2.1692(3)
1.9888(9)
1.9844(9)
2.4406(12)
2.4319(11)
2.121(3)
2.128(4)
2.042(3)
2.063(3)
2.166(1)
2.173(1)
1.931(4)
1.942(4)
2.1679(16)
2.1679(16)
1.939(4)
1.939(4)
Bond angles (°)
S1–Ni–S2
S1–Ni–N1
S1–Ni–N2
S2–Ni–N1
S2–Ni–N2
N1–Ni–N2
O1–Ni–S1
O1–Ni–S2
O2–Ni–S1
O2–Ni–S2
O2–Ni–N1
O2–Ni–N2
O2–Ni–O1
N1–Ni–O1
N2–Ni–O1
85.37(4)
89.31(9)
173.14(7)
172.28(9)
88.26(8)
97.3(1)
80.3(3)
83.8(2)
166.1(3)
83.6(2)
91.9(4)
96.00(12)
87.6(3)
102.5(3)
174.3(3)
86.7(3)
88.044(14)
88.72(3)
174.90(3)
175.00(3)
88.57(3)
95.77(4)
89.69(10)
83.83(9)
82.06(9)
89.78(9)
94.12(13)
97.78(13)
169.46(12)
91.84(12)
90.19(13)
91.32(4)
94.01(6)
89.9(1)
170.9(1)
170.3(1)
89.1(1)
88.3(2)
96.18(9)
86.06(10)
177.61(10)
174.96(10)
86.30(10)
96.32(14)
90.24(13)
163.74(11)
163.74(11)
90.24(13)
87.6(2)
97.0(4)
91.4(4)
85.8(3)
92.4(4)
170.3(4)
a Equivalent atoms generated by symmetry transformations.
program ORTEP-3 for Windows or CaChe-6 (Fujitsu Ltd, 2004)
was used89 for the structural diagrams.
(8) Å, c = 15.4674(13) Å. α = γ = 90°, β = 83.251(4)° and Z =
4. Rint = 0.1173. The structure was refined to R = 6.41% and Rw
= 13.83% (I > 2σ(I)). Pertinent crystallographic data is provided
in Table 1. Details are provided in SI (#4).†
Complex 2. The crystal used to solve complex 2 was a red
colored plate (BSC1a) with approximate dimensions 0.2 × 0.2 ×
0.02 mm3. A total of 10794 independent reflections were col-
lected at 200 K, and final refinement was based on 6246
observed reflections (I ≥ 2σi). Sum formula is NiC11H24N2S2.
FW is 307.15. Crystals of 2 form in the monoclinic space group
P21/n with cell dimensions a = 10.5547(10) Å, b = 12.3943(11)
Å, c = 11.5831(11) Å. α = γ = 90°, β = 111.126(5)°, and Z =
4. Rint = 0.087. The structure was refined to R = 4.40% and Rw =
10.22% (I > 2σ(I)). Pertinent crystallographic data is provided in
Table 1. Details are provided in SI (#2).†
Complex 8. The crystal used to solve complex 8 was a blue-
purple colored plate (BTW17o) with approximate dimensions
0.30 × 0.02 × 0.02 mm3. A total of 3752 independent reflections
were collected at 298 K, and final refinement was based on 3556
observed reflections (I ≥ 2σi). Sum formula is NiC10H19N2O12.
FW is 417.96. Crystals of 8 form in the orthorhombic space
group Pna21 with cell dimensions a = 14.669(5) Å, b = 16.306
(5) Å, c = 7.034(2) Å. α = γ = β = 90°, and Z = 4. Rint = 0.029.
Flack parameter = 0.047 (8). The structure was refined to R =
3.68% and Rw = 9.41% (I > 2σ(I)). Details are provided in SI
(#5).†
Complex 3. The crystal used to solve complex 3 was a blue
block (BTW5s) with approximate dimensions 0.21 × 0.32 ×
0.37 mm3. A total of 7086 independent reflections were col-
lected at 298 K, and final refinement was based on XYZ cen-
troids of 6974 reflections above 20 σ(I) with 2.320° < θ <
28.167°. Sum formula is NiC17H40I2N4O4S2. FW is 741.16.
Complex 10. The crystal used to solve complex 10 was a red-
pink colored plate (BSC1o) with approximate dimensions 0.01 ×
0.12 × 0.22 mm3. A total of 1650 independent reflections were
collected at 298 K, and final refinement was based on XYZ cen-
troids of 915 reflections above 20 σ(I) with 2.248° < θ <
24.842°. Sum formula is NiC10H22N2S2. FW is 293.13. Crystals
of 10 form in the orthorhombic space group Pbcn with cell
dimensions a = 9.487(6) Å, b = 7.862(5) Å, c = 18.145(13) Å. α
= β = γ = 90°, and Z = 4. Rint = 0.0863. The structure was
refined to R = 6.32% and Rw = 14.51% (I > 2σ(I)). Pertinent
crystallographic data is provided in Table 1. Details are provided
in SI (#6).†
ˉ
Crystals of 3 form in the triclinic space group P1 with cell
dimensions a = 10.4046(12) Å, b = 10.9123(12) Å, c = 13.8838
(16) Å, α = 87.671(2)°, β = 78.132(2)°, γ = 72.023(2)° and Z =
2. Rint = 0.013. The structure was refined to R = 4.90% and Rw =
13.86% (I > 2σ(I)). Pertinent crystallographic data is provided in
Table 1. Details are provided in SI (#3).†
Complex 6. The crystal used to solve complex 6 was a blue
colored plate (BC39) with approximate dimensions 0.15 × 0.15
× 0.05 mm3. A total of 3113 independent reflections were col-
lected at 293 K, and final refinement was based on 2038
observed reflections (I ≥ 2σi). Sum formula is NiC13H28I2N4O2-
S2. FW is 649.02. Crystals of 6 form in the monoclinic space
group P21/n with cell dimensions a = 11.2811(9) Å, b = 12.8465
C. Physical measurements: quenching experiments
UV-visible absorbance measurements (UV-vis) were obtained on
a HP 8453 Diode Array Spectrometer, or a Cary 4000 UV-vis
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Spectrophotometer. All experiments were performed at room
temperature. The fluorescence emission spectra were obtained
using a PE LS50B Spectrometer. Fluorescence spectra were nor-
malized and then corrected for wavelength dependent sensitivity
using a calibration curve generated using five spectral fluor-
escence standards (BAM-F001 to F005, Sigma-Aldrich).90 For
each series, the protein (or free Trp) concentration was held con-
stant at 1.0 × 10−5 M and the concentration of each Ni(II)
complex was varied (0.0 μM to 21.0 μM) through the preparation
of individual samples. The excitation wavelength was 280 nm,
emission wavelength was 345 nm and excitation and emission
slit widths were 3.5 nm each. The dynamic quenching constants
were determined using the Stern–Volmer equation:67,71
conditions, such as temperature and protic solvent con-
ditions..95,96 One way to prevent such oligomerizations is to use
bulky and/or aromatic substituents and/or chelate ligands that
readily undergo template reactions. The latter method was used
to synthesize four of the complexes reported herein. The syn-
thesis of each of the low spin neutral complexes 1, 2, 9, and 10
are analogous (SI #1†). Complexes 1 and 9 have been previously
synthesized, and crystal structures reported..95,97,98 All character-
ization data, including ESI-MS spectra and elemental microana-
lysis are consistent with the formulations and structures given for
each complex (see SI #1†). Selected bond lengths and angles for
1, 2, 9, and 10 are given in Table 1. The molecular structures of
each of these four complexes reveal discreet mononuclear units,
with no intermolecular Ni–S interactions.
F0
Complexes 1.97 and 9.98 have been previously studied by X-
ray crystallography. Complexes 2 (Fig. 2) and 10 (Fig. 3) are
novel. A comparison of the crystal structures of 1 and 2, and that
of 9 and 10 show that there is very little difference in bond
lengths and angles when a methyl group is placed on the carbon
alpha to the thiolates. A slight opening of the S1–Ni–S1 angle
and a slight closing of the N1–Ni–N2 angle is observed. The
most striking difference between complexes 1 and 9 is in the N-
methyl groups: in 1 they are cis in configuration, (axial to the
N1, N2, C3, C5 plane) whereas in 9 they are trans (to the N1,
N2, C4, C5 plane), a result of the steric requirements of a five-
membered ring, and the tetrahedral requirement of the nitrogen
atoms. The S–Ni–S bond angles in complexes 1 and 2 are more
acute (by about 9°) compared to the corresponding angles in
complexes 9 and 10. Conversely the N–Ni–N angle in com-
plexes 1 and 2 are more obtuse (by about 9°) compared to the
corresponding angles in 9 and 10. These angular differences
would be expected given the smaller chelate bite angle of the
‘ethyl strap’ in complex 9.
¼ 1 þ KSV½Qꢀ
ð1Þ
F
F0 and F correspond to the fluorescence intensities of the protein
without quencher and in the presence of the quencher, respect-
ively. [Q] is the concentration of the quencher and KSV is the
Stern–Volmer quenching constant. Also, KSV is related to the
lifetime of the system according to eqn (2):67
KSV ¼ kqτ0
ð2Þ
kq is the bimolecular quenching constant and τ0 is the lifetime
of the unquenched fluorophore. τ0 is short for Trp alone (2.7 ns)
but longer in a protein environment (9.8 ns).91–93 The static
quenching constant, also known as the binding constant, Ka, and
number of binding sites (n) between the protein and metal com-
plexes were calculated using eqn (3):67,94
ꢀ
ꢁ
F0 ꢁ F
log
¼ logKa þ nlog½Qꢀ
ð3Þ
F
The modified Stern–Volmer plot is useful when studying a
protein that has Trp in different environments, for example
buried residues versus residues at the surface, as is the case with
both BSA and lysozyme.66,67 In such cases the Stern–Volmer
plot will have a downward curve indicating that some of the flu-
orescence is not accessible. In this study, the modified Stern–
Volmer analysis was not relevant as the complexes were quench-
ing the proteins and free Trp via static quenching.
Results and discussion
The series of small water soluble Ni(II) complexes (Fig. 1) rep-
resents molecules that vary in charge (neutral, cationic, anionic),
spin state (low, high), and degree of hydrophilicity by virtue of
the nature of the ligands.
A. Synthesis and characterization of the nickel complexes
Complexes 1, 2, 9, and 10. These four Ni(II) compounds are
cis diamino dithiolate square planar complexes. Sulfur contain-
ing ligands readily form [NiL2]2− type four coordinate com-
plexes in the presence of a large excess of dithiolato ligand (L2).
At lower ligand concentrations, the propensity of thiolato-S to
form bridges between metal centers gives rise to multinuclear
species, an inconvenience that is also dependent on reaction
Fig. 2 ORTEP plot of complex 2, with ellipsoids at 50% probability.
The H atoms have been excluded for clarity. Full structural details are
given in SI (#2).†
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Fig. 5 ORTEP plot of complex 6, with ellipsoids at 50% probability.
The H atoms and the two iodide counterions, have been excluded for
clarity. Full structural details are given in SI (#4).†
Fig. 3 ORTEP plot of complex 10, with ellipsoids at 50% probability.
The H atoms have been excluded for clarity. Full structural details are
given in SI (#6).†
The most notable structural difference between the iodoaceta-
mide derivatives is that complex 6 contains Ni(II) with cis-cis-cis
geometrical N2S2O2 donor ligands, whereas complex 3 contains
cis-cis-trans geometry with the O’s being trans: these large
atomic rearrangements, in going from 4 to 6 coordinate avoids
cis configuration of the N-methyl groups. The expansion of the
coordination sphere also leads to expansion of all Ni-N and Ni–
S bond lengths. The most pronounced is that for the Ni–S bonds,
which (on average) goes from 2.17 Å to 2.43 Å. This is similar
to that observed by others in the literature for a similar Ni(II)
environment..99
Complexes 3 and 6. The alkylation of complex 1 or 2 with
two equivalents of iodoacetamide gives complex 6 or 3, respect-
ively (see SI #1†). These alkylated complexes are high spin (S =
1) dications, blue to purple in color, with pseudo octahedral
Ni(II) centers, each with a N2S2O2 donor ligand set, and iodide
counterions. The iodoacetamide derivatives were readily crystal-
lized (as beautiful gems) from MeOH/MeCN solutions and are
very stable in the solid state. ORTEP plots of the crystal struc-
tures are shown in Fig. 4 and 5.
Complexes 4 and 7. Both of these neutral high spin com-
plexes were synthesized according to published pro-
cedures..100,101 These Schiff base complexes are hydrolytically
stable over a wide pH range, due in part to the 5-6-5 membered
chelate rings..100,102,103 The only significant difference between
complexes 4 and 7 is the presence of a hydroxyl group on the
second carbon of the NN′ propyl strap. It is expected that the
additional feature will enhance the polar solubility of complex 7.
Complexes 5, 11, and 12. The synthesis of each of these high
spin Ni(II) amine/ammine complexes is well documented. Each
of the complexes is very soluble in polar solvents, intensely
colored, and make for an interesting comparison with the other
complexes in this series. Complex 5 is neutral, and complexes
11 and 12 are dications in solution.
Complex 8. The key role of the Ni(II) complex of EDTA in
our studies is that it is anionic. The complex is stable and X-ray
diffraction studies suggest that the complex in the solid state is a
quinquedentate octahedral complex, with one non-complexed
protonated acetate, and a water molecule bonded to the Ni. The
structure, as shown in Fig. 6, is similar to that reported in early
literature,.104 however some differences are noticeable [as would
be expected 52 years later], and for that reason an updated
Fig. 4 ORTEP plot of complex 3, with ellipsoids at 50% probability.
The H atoms and the two iodide counterions have been excluded for
clarity. Full structural details are given in SI (#3).†
This journal is © The Royal Society of Chemistry 2012
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Fig. 7 Fluorescence spectra of BSA (1.0 × 10−5 M) in the presence of
different concentrations of complex 2.
A typical plot of Trp fluorescence quenching with increasing
complex concentration is shown in Fig. 7. In this case (BSAwith
complex 2), the fluorescence decreases by approximately 26% at
the highest complex concentration used. The fluorescence
quenching ranged from 0–56% in this study (Table 3), with the
upper limit being slightly lower than the 60% quenching
observed in our recently reported study with a Cr(III) complex.76
Two common mechanisms for fluorescence quenching are
dynamic quenching and static quenching. In order to determine
the type of quenching taking place, two approaches are available
and commonly used. The first involves a temperature-dependent
study, as diffusion increases at higher temperatures, and colli-
sional quenching and subsequent dissociation of weakly bound
complexes leads to a decrease in static quenching. In the second
approach, as used in this work, the linearity of the Stern–Volmer
plot is interpreted. The Stern–Volmer plot of F0/F against [Q]
should be linear (eqn (1)), and this is usually the case for gas-
phase and liquid-phase systems where either kq or τ0 is large. In
the case of the proteins and free Trp, τ0 is small (10−8 to 10−9 s),
which leads to Stern–Volmer plots that curve upwards.67,76
Sample plots for BSA quenching by complex 4 are given in
Fig. 8. The dynamic quenching plot (Fig. 8B) is curving slightly
upward even at low complex concentrations. The deviation from
linearity is more prominent at higher complex concentrations as
illustrated in the case of lysozyme and complex 4 (Fig. 9). For
Fig. 6 ORTEP plot of complex 8 monoanion, with ellipsoids at 50%
probability. The H atoms and the Na+ counterion have been excluded for
clarity. The ligand labeled O9 is water. Full structural details are given in
SI (#4).†
crystal structure is presented here. A careful study of the packing
relations between molecules strongly indicates that hydrogen
bonding contributes significantly to the stability of the crystal.
B. Fluorescence quenching by nickel complexes
The fluorescence quenching observed in our studies is a result of
the interaction between the ligand that is bound to the Ni
complex, the Trp residue and its immediate vicinity in the
protein. Table 2 lists the Trp environment in the proteins studied.
The extent of quenching observed varied with protein and with
complex, and can be narrowed to some key factors: (i) the hydro-
phobicity of the ligand, (ii) presence or absence of complex
charge, and (iii) the nature of the amino acids surrounding the
Trp. While it has been reported in the literature that Ni ions can
bind to BSA and form either square planar or octahedral com-
plexes,28 the quenching observed is dependent on the nature of
the ligand.
Table 2 Trp environments in BSA, HSA and lysozyme.105 Tyr residues are partially hydrophobic
Surrounding amino acid residue(s)
Protein
Trp residue(s)
Positively charged and amphipathic
Negatively charged
Polar-uncharged
Hydrophobic
BSA
Trp-134
Trp-212
Trp-214
Trp-28
Trp-62,63
Trp-108
Trp-111
Trp-123
—
—
—
Lys
Arg
—
Arg
—
Glu
—
—
—
—
—
Glu
—
Ser
—
—
Asn, Thr
Tyr, Val
Tyr, Leu, Phe
Tyr, Val
Val
HSA
Lyso
Ser
Asn, Gln, Thr
Asn
—
Val
Ile
2726 | Dalton Trans., 2012, 41, 2720–2731
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Table 3 Percent decrease in fluorescence intensity of fluorophore (1.0 × 10−5 M) after addition of highest amount of quencher (21 μM)
Nickel complexes
Protein
1
2
3
4
5
6
7
8
9
10
11
12
BSA
HSA
Lyso
Trp
17.2
20.3
17.2
22.1
0
26.3
23.7
21.1
No
No
13.0
No
No
+2
49.4
54.5
55.6
55.9
0
No
No
No
No
0
No
No
No
No
+2
No
No
No
No
No
−1
43.4
48.3
43.3
48.0
0
31.3
28.4
41.3
42.0
0
No
No
No
No
+2
No
No
No
No
+2
40.6
19.3
36.5
0
Charge on complex
0
Fig. 8 Stern–Volmer plots for (A) static quenching (R2 = 0.993) and (B) dynamic quenching of BSA by complex 4 (R2 = 0.9884).
Fig. 9 Dynamic quenching plot for lysozyme (1.0 × 10−5 M) quenching by complex 4. (A) The fluorescence of the protein is fully quenched when
[4] approaches 120 μM. (B) Expansion of the enclosed area in A. Deviation from linearity is evident at complex concentrations less than 21 μM.
static quenching, the binding constant (Ka) for the formation of
an adduct between the Ni(II) complexes and BSA was deter-
mined using a double logarithmic plot (Fig. 8A). The binding
site values (n) were also determined from the slope using
eqn (3).
surprisingly consistent with our recent study involving a Cr(III)
complex where a value of 1.8 × 1013 M−1 s−1 was determined.76
These large Stern–Volmer constants strongly suggest that static
quenching is predominant in these systems. Even though the evi-
dence points towards static quenching, the KSV and kq values
merit some discussion: The kq values provide a sensitive
measure of the exposure of Trp residues in proteins. A low kq
value indicates that the protein is sterically shielding the Trp
from the solvent.13,67,74 In the systems we have studied, that
Another important consideration is that for dynamic quench-
ing, the maximum value of kq is normally in the range of 2.0 ×
1010 M−1 s−1 28,73,105
However, in this study, we found values of
.
kq that are of the order of 1012 to 1013 M−1 s−1, which are
This journal is © The Royal Society of Chemistry 2012
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clearly is not the case as the kq values are much greater. The kq
values reported in this work are in good agreement with values
reported in the literature for BSA-baicalin (∼1.7 × 1013 M−1
s−1), BSA-kaempferol (∼8.5 × 1012 M−1 s−1) and BSA-[Cr
(salprn)(H2O)2]+ (∼1.9 × 1012 M−1 s−1).13,74,106 Fluorescence
quenching is often a result of energy transfer from a donor (Trp)
to an acceptor (metal complex).74 However, in all the systems
and combinations studied, the quenching of Trp fluorescence is
static and Forster’s long-range energy transfer is not taking
place. Also, the spectral overlap between Trp emission and
complex absorption is negligible (Fig. 10) compared to other
systems.45,72,73,76,106 The presence of an overlap between the
emission band of the donor and the absorption band of the
acceptor is a requirement for FRET. Therefore, the Ni(II) com-
plexes are quenching the fluorescence via a thermodynamically
favorable (ΔG < 0) interaction with a high binding constant.
hydrogen-bonding interactions resulting from the presence of an
OH group in 7. The high binding constants (Ka) correlate with
the negative values obtained for ΔG, confirming that binding is
spontaneous. Similar values have been observed in other studies
involving BSA, HSA and lysozyme.45,46,72,73,76,106–108 The ΔH
value is zero for hydrophobic interactions, and so ΔS is positive.
ΔG values range from −5 to −40 kJ mol−1 when one binding
site is involved (n = 1), and previous studies76 have yielded
values in the 15 to 35 kJ mol−1 range. When more than one
binding site is involved, the association is even more thermody-
namically favorable, with ΔG from −40 to −60 kJ mol−1
.
i. BSA and HSA. Both serum albumin proteins were
quenched by complexes 1, 2, 9 and 10. These complexes are
similar in structure, with what appear to be subtle differences.
The binding constants, Ka, for these four complexes were of the
order of 103 and higher. The fluorescence quenching by complex
9 was the greatest in this group (Table 3, 43% for BSA and 48%
for HSA) and the Ka values were as high as 107, indicating
strong binding by this particular complex to the Trp region of the
proteins (Table 4). Complex 9, with its 5-5-5 membered chelate
rings, is slightly smaller than complexes 1, 2 and 10 thereby
lending itself to a slightly stronger interaction with the protein.
The quenching by the slightly larger complex 10 was signifi-
cantly lower at 30%, with Ka values of the order of 104–105
M−1. For complex 2, the quenching was about 25% in both
cases, and the Ka values were of the order of 104 to 105 M−1. A
correlation between complex size and the ability to quench and
bind strongly is evident.
C. Nature of the interaction between the proteins and nickel
complexes
The proteins used in this study have complex structures and
therefore many types of interactions are possible between the
protein and a small complex or molecule. Van der Waals, electro-
static, hydrophobic and hydrogen bonding interactions are all
possible.106 The resulting fluorescence quenching of Trp occurs
only when there is interaction with the hydrophobic ligands, and
the interaction is either promoted or stabilized by the surround-
ing amino acid residues (Table 2).
The most noticeable correlation from data in Table 3 is that
small neutral alkyl Ni(II) complexes (1, 2, 9 and 10) quench flu-
orescence better than the bulky charged complexes, such as 3, 6,
and 8. The spin state (high spin for the hexacoordinate, and low
spin for the tetracoordinate complexes) appears to play little part
in the interaction. The hexacoordinate complexes studied are for
the most part charged, and only complexes 4, 5 and 7 are
neutral. Of these three neutral complexes, 4 and 7 possess hydro-
phobic aromatic ligands and are amongst the strongest fluor-
escence quenchers. The significant decrease in quenching
observed between BSA and lysozyme with complex 7 is due to
A close examination of the immediate environment of each
Trp (Table 2) indicates that there is at least one hydrophobic, par-
tially hydrophobic or amphiphilic amino acid in the immediate
vicinity. In both BSA and HSA, the partially hydrophobic Tyr is
buried in the hydrophobic core. Hydrophobic amino acids such
as Leu and Val are capable of recognizing and binding hydro-
phobic ligands. The Tyr residue is also capable of interacting
with ligands via stacking interactions, which appears to be the
nature of the interaction with the pyridinium rings in complexes
4 and 7. In the case of complex 4, the fluorescence quenching is
the greatest (> 49%) and the binding constants are of the order
of 105 M−1. Interestingly, HSA showed a strong association with
complex 7 (Ka ∼105) but BSA did not. The ligand in complex 7
differs from that in complex 4 in only one regard, it contains an
OH group. Trp-134 in BSA has a polar amino acid (Ser) in its
vicinity and so it is possible that an H-bonding interaction
occurs between this residue and complex 7, in addition to the
Table 4 Summary of binding constant, Ka, binding site number, n, and
ΔG for BSA and HSA
BSA
HSA
Complex Ka/M−1
n
ΔG/kJ mol−1 Ka/M−1
n
ΔG/kJ mol−1
1
2
3
4
5
7
9
10
3.2 × 104
2.7 × 105
—
1
1
—
1
—
—
−26
−31
—
−31
—
5.7 × 109
1.4 × 104
9.1 × 104
2.2 × 105
—
2
1
1
1
—
−56
−24
−28
−30
—
3.2 × 105
—
—
—
1.8 × 105
1
−30
Fig. 10 Overlap of Trp emission of 1.0 × 10−5 M Trp (solid line) with
the absorption of 1.0 × 10−5 M complex 1, showing negligible absor-
bance of the complex in the region of Trp fluorescence emission.
3.8 × 107 1.5 −43
1.3 × 107 1.5 −41
5.8 × 105
1
−33
2.4 × 104
1
−25
2728 | Dalton Trans., 2012, 41, 2720–2731
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H-bonding involving Trp. The H-bonding interaction is stronger
than any stacking interaction that would be expected between
Tyr (or Trp) and the pyridinium group(s) in the complex. The H-
bonding between the OH group in the complex and the Ser (and/
or Trp) residue is also stronger than hydrophobic interactions
promoted by the Leu and Val residues that are in the vicinity of
Trp-134. This rationale explains the absence of fluorescence
quenching and absence of binding of complex 7 to BSA. On the
other hand, HSA does not have a polar amino acid near its Trp-
214 so the hydrophobic interactions predominate, leading to flu-
orescence quenching.
HSA weakly interacts with the neutral complexes 1 and 2
(about 20% quenching), very weakly with the cationic complex
3 (13% quenching), and not at all with complex 6. These weak
or non-existent interactions with 3 and 6 are most likely due to
the presence of NH2 groups in the respective ligands, the inter-
action with Trp-214, and the influence of the nearby tyrosine
residues in HSA. Once again, H-bonding is possible between the
NH2 groups and the amino acids. No interaction was detected
for 3 and 6 with BSA due to the presence of more hydrophobic
groups, and of course the lower hydrophobicity of the ligands in
these complexes.
No fluorescence quenching was observed for any proteins
with complexes 11 and 12. The ligands on complexes 11 and 12
are strongly hydrophilic, and therefore not exhibiting the type of
interaction that would lead to quenching of protein fluorescence.
In general, interaction between complexes and proteins occurred
only with complexes containing hydrophobic ligands. This
observation underscores the nature of the Trp residue, and the
importance of the immediate vicinity being occupied by one or
more hydrophobic residues, or at least partially hydrophobic
amino acid residues.
which leads to a disruption in binding and quenching. There was
no change in fluorescence intensity when 3.0 μM of complex 4
was added but further addition of the complex resulted in signifi-
cant static quenching over the 3.0 to 21 μM range (Fig. 9). The
high fluorescence quenching resulted in a higher than average
binding constant (∼1010 M−1) and a binding site number of 2.
iii. Free tryptophan. Free Trp showed very similar behavior
towards the Ni(II) complexes as the three proteins. The inter-
action was strongest with neutral complexes 1, 4, 7, 9 and 10
(Table 5). The absence of quenching with complex 2 is anoma-
lous, and could be attributed to the steric effects resulting from
the slightly larger size of the ligand when compared to the alkyl
ligands in complexes 1, 9 and 10. The interaction with 7 is sig-
nificantly weaker due to the presence of the OH group on the
ligand in the complex. This observation opens up the possibility
of non-hydrophobic interactions taking precedence over hydro-
phobic interactions between the Trp and the pyridinium groups
in the complex. This is similar to what was observed with BSA
where there was no interaction with 7, but a strong interaction
with 4.
Our data suggests that in most of the systems studied, one
binding site is present in each, the exceptions include all the
interactions with lysozyme (except with complex 10), HSA with
complex 1 (n = 2), HSAwith complex 9 (n = 1.5), and BSAwith
complex 9 (n = 1.5). A binding site number of 1 is typical when
static quenching of proteins occurs as a result of hydrophobic
interactions with transition metal complexes and other mol-
ecules. The presence of two binding sites in lysozyme is not
unusual as there are six Trp residues in the protein, and complex
4 can associate with the different Trp residues via stacking inter-
actions through the pyridinium groups. The small size of com-
plexes 1, 2 and 9 is also an important factor, allowing these
complexes to squeeze into additional binding sites. Complex 9 is
the smallest of these complexes, and is found to bind more
strongly to both BSA and HSA compared to any of the other
complexes, therefore a higher binding site number is not unex-
pected. BSA has two and HSA has only one Trp residue, and so
the presence of two binding sites in HSA raises the possibility
that multiple small quencher molecules may be involved since
they would have easy access to the Trp residue via hydrophobic
interactions.
ii. Lysozyme. Lysozyme fluorescence was quenched by
complexes 1, 2, 4, 7, 9 and 10. These are all neutral complexes,
with hydrophobic ligands. Significant quenching was observed
with complex 4. Given that lysozyme has six Trp residues, a
variety of interactions are possible between the protein and the
Ni(II) complexes. Trp-28, Trp-62, Trp-63 and Trp-111 are close
to amphiphilic amino acids like Lys and Arg, while Trp-108 and
Trp-123 are close to hydrophobic Val and Ile, respectively. The
hydrophobic interactions are the most important. In terms of
binding constants, the interaction between lysozyme and
complex 4 was exceptionally strong while the interaction with 7
was the weakest (Table 5). The ligand in complex 7 differs from
that in 4 in only one regard in that it contains an OH group,
Quantum chemical computational studies using ab initio
methods (Gaussian09) are currently under way to determine the
energetic contribution of the interactions between the Trp resi-
dues and the surrounding amino acids in HSA, BSA, and lyso-
zyme, with these Ni(II) complexes.
Table 5 Summary of binding constant, Ka, binding site number, n, and
ΔG for lysozyme and free Trp
Conclusions
We have synthesized and fully characterized a series of Ni(II)
complexes. Fluorescence quenching studies with albumins and
lysozyme proteins clearly suggest that the Ni(II) complexes fea-
turing hydrophobic ligands exhibit strong and favorable inter-
actions. These interactions take place between the hydrophobic
pocket of the protein and the ligand. The poorest interactions are
with charged bulky Ni(II) complexes. Static quenching was
found to be the predominant form of quenching taking place in
these systems, and generates high binding constants. The small
Lysozyme
Complex Ka/M−1
Free Trp
n
ΔG/kJ mol−1 Ka/M−1
n
ΔG/kJ mol−1
1
2
4
7
6.7 × 107
5.1 × 107
1.6 × 1010
—
2
2
2
—
−45
−44
−58
—
1.4 × 103
—
1
—
1
1
1
−18
—
−30
−15
−28
−31
2.0 × 105
4.6 × 102
9.8 × 104
2.4 × 105
9
10
5.4 × 106 1.5 −38
2.8 × 105
1
−31
1
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