D.E. Lynch et al. / Dyes and Pigments 94 (2012) 393e402
395
(
4H, t, J ¼ 6.0, ArH), 8.33 (4H, d, J ¼ 7.0, ArH), 8.46 (4H, d, J ¼ 7.0,
tetraacetic acid (EDTA), glacial acetic acid, glycerol, hydrochloric
acid (HCl), human serum albumin (HSA), mercaptoethanol, meth-
anol, polyethyleneglycol (PEG), sodium acetate, sodium dode-
cylsulfate (SDS), sucrose, 1,2-bis(dimethylamino)ethane (TEMED),
and 2-amino-2-hydroxymethylpropane-1,3-diol (Tris) were
purchased from SigmaeAldrich and were used as received without
further purification. 40% Acrylamide solution, 1 M TriseHCl buffer
solutions (pH 8.5 and pH 6.0), 20% SDS solution, 10% ammonium
persulfate (APS) solution, and a premixed 10ꢂ TriseglycineeSDS
Laemmli running buffer were all supplied by Scie-Plas and stored
þ
þ ꢀ
ArH), 9.85 (2H, s, ArH). ES : 180.2 (C13H10N ; calc.180.2). ES : 319.5
(
2
ꢀ
C
32
34
H N
2
O
8
S
2
; calc. 638.8).
2
.1.6. Bis-piperidinium 2,4-bis-(3,3-dimethyl-(1-propan-3-
sulphonate)-2-indolinylidenemethyl)cyclobutene-1,3-diolate (2e)
Same procedure as for 2b except: piperidinium (150 mg,
1
.76 mmol). Upon cooling, a metallic green powder was collected in
vaccuo, washed with petroleum ether (60/40), and used without
further purification. Yield: 250 mg (35%). UV/Vis (H O) max (log ):
max: 1580 (CeO). H NMR (400.13 MHz, DMSO-
2
l
3
1
ꢁ
6
d
CH
3
24 (5.43). IR (KBr)
: 1.61 (4H, m, CH
), 2.09 (4H, quintet, J ¼ 7.3, CH
n
at 4 C when not in use.
)
d
2
), 1.71 (8H, quintet, J ¼ 5.7, CH
2
), 1.72 (12H, s,
ProteMix Protein Standard containing 12 protein fractions1
(including two insulin fractions) with a molecular weight ranging
from 220 kDa (Myosin from Oryctol. cuniculus muscle tissue) to 3.4
and2.5kDa(InsulinfromBostauruspancreatictissue)waspurchased
6
ꢀ
3
2
), 2.61 (4H, t, J ¼ 7.0, CH
2
SO
3
),
.05 (8H, t, J ¼ 5.8, NCH
2
), 4.27 (4H, t, J ¼ 7.6, NCH
2
), 5.85 (2H, s,
CH]), 7.15 (2H, t, J ¼ 7.5, ArH), 7.33 (2H, td, J ¼ 7.6, J ¼ 1.2, ArH), 7.42
þ
ꢁ
(
2H, d, J ¼ 8.0, ArH), 7.45 (2H, d, J ¼ 7.5, ArH), 8.22 (4H, bs, N H).
from Anamed andwas storedat ꢀ18 C before beingwarmed to room
ꢀ
2ꢀ
2
ES : 319.4 (C32
H
34
N
2
O
8
S
; calc. 638.8).
temperature for use, as it was pre-prepared in its own sample buffer.
A ‘homemade’ protein ladder solution was prepared using HSA
(80 kDa), BSA (66 kDa), bovine carbonic anhydrase (29 kDa) and
insulin (3.4 and 2.5 kDa; from bovine pancreas) in water at twice the
2
.1.7. X-ray crystallographic analysis
Single crystals of the four squaraine dyes 2ae2d were obtained in
each case by the slow evaporation of a dilute solution of each dye in
desired concentrations (400 mg/mL) before preparation in sample
chloroform. Crystallographic data, for 1, was collected on an
buffer. Relative protein concentrations were determined individually
using Lowry assays [36], and a correction factor applied to equalize
the protein concentrations in the final solution. Rat liver mitochon-
drial supernatants were sourcedat Coventry Universityandprepared
by lysis followed by centrifugal separation in 0.25 M sucrose/20%
PEG, and were then dialysed to remove the salt content, before
preparing in sample buffer. Dialysis of the rat liver sample was per-
EnrafeNonius CAD-4 using monochromatized Mo-K
tion (
¼ 0.71073 Å), for 2c, was collected on a Bruker Nonius Kappa
CCD area diffractometer using monochromatized Mo-K X-ray
radiation (
a X-ray radia-
l
a
l
¼ 0.71073 Å) equipped with an Oxford Cryosystem low
temperature device, and for 2a, 2b, and 2d, was collected on a Bruker
SMART APEX2 CCD diffractometer using synchrotron radiation
0
0
(
l
¼ 0.6848 Å). All structures were solved by direct methods
formed in sections of dialysis tubing (8/32 ), cut to a length of 10 cm
and soaked in 250 mL water with 0.25 g EDTA for 16 h. These tubes
were then capped at one end with a plastic clip and 2 mL of rat liver
supernatant pipetted into them. The other end was then clipped and
SHELX97, and refined by full-matrix least-squares calculations.
Crystal data for 1: C14
a ¼ 10.767(4), b ¼ 12.782(2), c ¼ 11.891(6) Å,
H
23NO
5
S, Mw ¼ 317.39, monoclinic, P2
1
/c, Z ¼ 4,
ꢁ
b
¼ 103.88(2) ,
ꢀ
3
ꢀ1
ꢁ
D
2
calcd ¼ 1.327 g cm , T ¼ 298(2) K, F(000) ¼ 680,
m
¼ 0.224 mm
,
the tubes placed in 2 L of stirred water, for 24 h at 4 C with the water
923 reflections were collected, 2772 unique (Rint ¼ 0.2284), 845
observed (F > 4 (F)), 202 parameters, R ¼ 0.1920.
¼ 0.0788, wR
Crystal data for 2a: C50 /c,
, Mw ¼ 899.08, monoclinic, P2
being changed twice at regular intervals. The total protein concen-
trationofthelysedmitochondrialsupernatantswasthendetermined
using the Lowry assay, using BSA as a standard. The sample buffer
used to prepare all protein samples, except the ProteMix standard,
was made by adding 2.5 mL 0.488 M Triseacetate buffer (pH 8.0),
0.25 g of SDS, 2 mL (1.46 g) glycerol, 0.05 mL mercaptoethanol, and
0.5 mLof0.5% bromophenolblue solutionin a 10 mLvolumetric flask,
and then diluting to the mark with water. All samples and standards
were prepared for electrophoretic separation by vortex mixing in 1:1
proportions with the sample buffer followed by heating to at least
s
1
2
H
50
N
4
O
8
S
2
1
Z ¼ 2, a ¼ 13.3034(9), b ¼ 6.3940(4), c ¼ 27.4465(16) Å,
ꢁ
ꢀ
ꢀ3
b
m
¼ 110.965(3) , Dcalcd ¼ 1.370 g cm , T ¼ 120(2) K, F(000) ¼ 948,
1
¼ 0.184 mm , 13,214 reflections were collected, 3820 unique
int ¼ 0.0579), 2655 observed (I > 2 (I)), 291 parameters,
¼ 0.0452, wR ¼ 0.1023. Crystal data for 2b: C82
(R
s
R
1
2
92 8 24 4
H N O S ,
Mw ¼ 1701.92, monoclinic, C2/c, Z ¼ 4, a ¼ 44.255(2), b ¼ 6.6710(4),
ꢁ
1
ꢀ3
c ¼ 26.8252(14) Å,
F(000) ¼ 3584, ¼ 0.205 mm , 26,967 reflections were collected,
882 unique (Rint ¼ 0.0483), 5121 observed (I > 2 (I)), 639 param-
56 4 13 2
eters, R H N O S ,
b
¼ 90.732(2) , Dcalcd ¼ 1.428 g cm , T ¼ 120(2) K,
ꢀ
ꢁ
m
95 C for 3 min (water bath).
6
s
1
¼ 0.0743, wR
2
¼ 0.1855. Crystal data for 2c: C50
2.3. Spectroscopic measurements
Mw ¼ 985.11, triclinic, P ꢀı , Z ¼ 2, a ¼ 11.5003(12), b ¼ 14.1179(12),
ꢁ
c ¼ 14.8336(15) Å,
a
¼ 96.743(6),
b
¼ 98.322(4),
g
¼ 91.954(6) ,
Visible absorption spectra were recording on a Shimadzu UV-
ꢀ3
ꢀ1
D
4
calcd ¼ 1.384 g cm , T ¼ 120(2) K, F(000) ¼ 1040,
m
¼ 0.184 mm
,
1
650 UV/visible spectrometer. Molar absorptivity for each dye
ꢀ5
4,316 reflections were collected, 9235 unique (Rint ¼ 0.1053), 5504
observed (I > 2 (I)), 656 parameters, R ¼ 0.1594.
¼ 0.0961, wR
Crystal data for 2d: C58
derivative was determined by serial dilution from 1 ꢂ 10
M
s
1
2
standard solutions, using ten results for each calculation.
H
58
N
4
O
10
S
2
, Mw ¼ 1035.22, triclinic, P ꢀı , Z ¼ 1,
a ¼ 6.710(2), b ¼ 9.413(4), c ¼ 19.966(7) Å,
a
¼ 91.061(4),
2.4. Gel casting procedure
ꢁ
ꢀ
ꢀ3
b
¼ 93.003(4),
g
m
¼ 102.736(4) , Dcalcd ¼ 1.400 g cm , T ¼ 120(2) K,
1
F(000) ¼ 546,
¼ 0.177 mm , 12,308 reflections were collected,
(I)), 342 param-
¼ 0.4343. Crystallographic data (1 (CCDC
98555), 2a (CCDC 798554), 2b (CCDC 798556), 2c (CCDC 798557),
d (CCDC 798558)) have been deposited at the CCDC,12 Union Road,
The gels used in the sodium dodecylsulphate polyacrylamide gel
electrophoresis (SDS-PAGE) experiments were made using
6
720 unique (Rint ¼ 0.0328), 5294 observed (I > 2
s
eters, R
7
2
1
¼ 0.1477, wR
2
1
Molecular weight fractions (kDa): 220 (Myosin from Oryctol. cuniculus muscle
Cambridge CB2 1EZ, UK.
tissue), 116 (
Oryctol. cuniculus muscle tissue), 66.0 (Albumin from Bos taurus serum), 55.6
Glutamate Dehydrogenase from Bos taurus liver), 36.5 (Lactate Dehydrogenase
from Sus scrofa muscle tissue), 29.0 (Carbonic Anhydrase from Bos taurus erythro-
cytes), 20.0 (Trypsin inhibitor from Glycine max), 14.0 (Lysozyme from Gallus gallus
egg white), 6.1 (Aprotinin from Bos Taurus lung tissue), and 3.4 and 2.5 (Insulin from
Bos taurus pancreatic tissue).
b-Galactosidase from E. coli), 97.0 (Glycogen-Phosphorylase from
(
2.2. Materials
Bovine serum albumin (BSA), bromophenol blue, Coomassie
Brilliant Blue R-250 (CBB), 2,2 ,2 ,2 -(ethane-1,2-diyldinitrilo)
0
00 000