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4.4. Protein concentration (Bradford)
1E4 for ion trap MS scan and 5E5 for FTMS full scan, respectively.
Fourier-transform mass spectra were recorded from 100 to 1000 Da
at a resolution of 100.000 (at m/z 400), each scan consists from 30
transients.
Bradford test [23] was applied for the determination of pro-
tein concentration in solution. Calibration standards of different
dilutions of a BSA stock solution in water as well as the unknown
protein concentration was determined by adding 200 L Bradford
reagent to 800 L of diluted protein solution. Absorbance at 595 nm
was measured by means of a UVIKON 922 photometer (Kontron
Instruments, UK) after incubation for 5 min. The protein amount in
solution could then be determined from the calibration curve.
4.7. Thermostability studies of LbADH variants
The dissolved (0.1 mg/mL protein, 10 mM TEA, 1 mM MgCl2, pH
7.5) samples of LbADHwt and the LbADH G37D (each 100 L) were
incubated in Eppendorf tubes at 30–80 ◦C in a heating block (Stuart
SBH 130D). At specific time points sample tubes were taken and
the proteins assayed for residual enzymatic activity as described
above. The solid probes were prepared by lyophilization of 100 L
aliquots of the dissolved LbADHwt and LbADH G37D (0.1 mg/mL
protein, 10 mM TEA, 1 mM MgCl2, pH 7.5) in Eppendorf tubes. The
dry samples were equilibrated with a saturated salt solution of LiCl
for 24 h at RT to reach the water activity of 0.1, hermetically closed
and incubated at 30–80 ◦C in a heating block (Stuart SBH 130D). The
solid protein probes taken out at specific time points, cooled down
on ice for 1 min, re-dissolved in 100 L of ddH2O and analyzed for
the residual catalytic activity as described above. The inactivation
curves were described by linear, one or second-order exponential
decay models. Half-life of enzymatic activity was read from the
plots.
4.5. Cofactor thermostability studies
The stability of the dissolved cofactors was investigated in a
range of 30–70 ◦C in 50 mM TEA buffer, pH 7.2. LbADHwt enzy-
matic assay was used to measure the residual reducing activity of
the heat-treated NADPH samples, LbADH G37D was taken for the
NADH probes. Solid cofactor probes were directly weighed from
supplier’s material, were exposed to 50 ◦C for the time period of
0–24 days, then re-dissolved in 50 mM TEA buffer (pH 7.2) and
tested for absorption at 340 nm and reducing activity. Enzymatic
assay was performed as described above. The initial highest activity
value was taken as 100% and used to normalize the other data.
4.6. HPLC–MS analysis of structure elucidation of cofactor
degradation products
4.8. Fluorescence spectroscopy
Dissolved samples of NADPH and NADH were heated at 50 ◦C for
16 h in water, whereas the solid cofactor samples were heated at
95 ◦C for 16 h and subsequently analyzed by HPLC. HPLC–MS exper-
iments were carried out on an Agilent 1100 series binary HPLC
system (Agilent Technologies, Waldbronn, Germany), equipped
with a DAD (190–400 nm) and coupled with a 4000QTRAPTM linear
ion trap mass Spectrometer (Applied Biosystem/MDS SCIEX, Foster
City, CA, USA) equipped with a TurboIon spray source.
Thermal denaturation experiments were designed such that
heat-induced inactivation was completed in 30 min. Fluorescence
emission spectra of the proteins in 10 mM TEA, 1 mM buffer
MgCl2 at pH 7.2 were measured in a LS50B spectrofluorometer
(PerkinElmer) in the range 300–450 nm after excitation at 295 nm
with a speed of 120 nm min−1. Excitation and emission slits were
7.5 nm, respectively. The dissolved samples were measured directly
after heat treatment, whereas the solid ones were cooled down and
re-dissolved in the corresponding buffer.
The dissolved samples were investigated in the following man-
ner: 0.1 mg/mL solution of LbADHwt or LbADH G37D in 10 mM TEA,
1 mM MgCl2 buffer at pH 7.0 was divided into aliquots of 800 L
each, then directly heated at 50 ◦C for different time intervals and
subjected to analysis by fluorescence spectroscopy in correlation
with enzymatic activity.
Thermal decomposition of the solid enzyme samples was
accomplished as follows: 1 mg/mL solution of LbADHwt or LbADH
G37D in 10 mM TEA, 1 mM MgCl2 buffer at pH 7.0 were divided
into aliquots of 100 L each as before, then frozen at −20 ◦C and
lyophilized. After lyophilization solid protein probes were heated
at 80 ◦C, cooled down to room temperature, re-dissolved in 1 mL
of distilled water, and immediately tested for residual enzymatic
activity and fluorescence properties.
HPLC separation was achieved on a ZIC-pHILIC PEEK column
(SeQuant, Marl, Germany, 150 mm × 4.6 mm, I.D., 5 m particle
˚
diameter, 200 A pore size) and a pre-column (20 mm × 2.1 mm, I.D.)
filled with the same material. The isocratic elution was performed
with a mixture of acetonitrile and 50 mM ammonium carbonate
(pH 8.0) (70:30, v/v) at the flow rate of 0.5 mL/min (split ratio 1:1)
kept at 30 ◦C during analysis. The injection volume was 2 L. The
MS was operated in the positive and negative Enhanced MS (EMS)
mode scanning from 100 to 900 Da with a line ion trap (LIT) fill time
of 2 ms and a Scan Rate of 4000 Da s−1
.
The parameters used for all methods were optimized first per-
forming a Flow Injection Analysis (FIA) with NADH as a standard
and led to the following parameter settings: IS −4500 V, Decluster-
ing Potential (DP) −145 V, Curtain Gas (N2) 10 arbitrary units (au),
Source Temperature 650 ◦C, Nebulizer Gas (N2) 50 au and Heater
Gas (N2) 20 au. CE and Q3-Entry barrier were set to −5 V and 8 V,
respectively, to minimize fragmentation entering the LIT.
4.9. Static light scattering
For structure elucidation and confirmation of selected ions
highly resolved mass spectra were recorded using a ESI-LTQ-FT
Ultra (ThermoFisher Scientific, San Jose, CA, USA), equipped with
a 7 T supra-conducting magnet and coupled with the chip-based
micro-ESI system NanoMate (Advion BioServices, Ithaca, NY, USA).
The mass spectrometer, used in positive and negative mode, was
tuned and external mass calibrated following a standard procedure
for all voltages and settings with a calibration solution composed of
caffeine, the peptide MRFA and ultramark. Therefore, the settings of
the ion optics varied slightly from day to day. The transfer capillary
temperature was set to 175 ◦C.
Static light scattering measurements were performed using a
Perkin Elmer LS 50B fluorescence photometer at 450 nm ( 15 nm
slit). The scattering curve was obtained at a fixed angle of 90◦,
in a 400–500 nm range with a 5 nm slit and a scanning rate of
120 nm min−1. For a single spectrum, 3 scans were accumulated.
The spectra were corrected for the buffer base line. The scattering
light intensity was determined at 450 nm. For the easier interpreta-
tion of results, the scattering light intensity was normalized to that
one of the respective intact enzyme. Experiments with the previ-
ously heat incubated LbADHwt and LbADH G37D were carried out
at room temperature. To study the dissolved samples, 0.1 mg/mL
solution of the LbADHwt or the LbADH G37D in 10 mM TEA, 1 mM
MgCl2 buffer at pH 7.5 was divided into 800 L aliquots, heated
Hand cut fractions from the analytical HPLC were put in a
96-well plate (zero-carryover) and sprayed continuously by the
NanoMate source. The automatic gain control (AGC) was set at