evidence of H/D exchange between LDH and
deuterium solvent in the frozen state is shown in Fig. 2. In
Fig. 1 Two protocols used for
HX labeling (Protocol A) in the
frozen state, and (Protocol B) after
subjection to F/T cycles. The time
for each step is designated
underneath.
1182 Zhang, Qi, Singh and Fernandez
this experiment, 0.1 mg/ml LDH was frozen in 90%
deuterium buffer using liquid N2, and then stored in a
−10°C freezer for a period of time ranging from 10 min up
to 4 days. The degree of deuterium labeling was measured
by the mass increase relative to unlabeled LDH by mass
spectrometry. As shown in Fig. 2, the molecular mass of
LDH increased with incubation time in the −10°C freezer,
demonstrating there was continuous incorporation of
deuterium into LDH within the experimental time range
in the frozen state. The deuteration level calculated by
equation (1) increased from 21.8% to 68.3% with the
labeling time increasing from 10 min to 4 day. For studies
under variable protein and solution conditions, an intermediate
incubation time of 12 h at −10°C was selected for
LDH conformation analysis in the frozen state for two
reasons. First, the observed LDH mass increase from
10 min to 12 h in Fig. 2 suggested that a significant
fraction of deuterium labeling was achieved in the frozen
sample state during the 12 h incubation, which reduced
the effect of H/D exchange in the solution state following
LDH sample and D2O labeling buffer mixing. Second, the
deuteration level of LDH after 12 h incubation was not
particularly high (38.6%) relative to the fully unfolded
control (100%), and it favored the resolution of labeling
peaks for native and partially unfolded LDH in the frozen
state shown below.
Selasa, 27 Desember 2011
LC-MS for HX Measurements
In both HX protocols in Fig. 1, the same experimental
setup was used for mass spectrometry analysis. For whole
molecule HX analysis, after quenching of H/D exchange
with 50 mM citric buffer (pH 2.5, containing 6 M
guanidine HCl), LDH sample was immediately loaded into
a sample loop. An isocratic pump delivered the LDH
sample in the loop to a peptide trapping column (1 mm
ID×8 mm, catalog No. TR1/25108/01; Michrom Bioresources,
Auburn, CA) for desalting. After 6 min desalting,
a short gradient of acetonitrile (ACN) from 25% to 90%
over 7 min (50 μL/min by Surveyor MS Pump, Thermo,
San Jose, CA) was used to elute LDH from the trapping
column and deliver it to the electrospray ionization ion-trap
mass spectrometer (LTQ, Thermo Electron Corporation,
San Jose, CA).
In the peptide level HX analysis, an in-line proteolytic
digestion was incorporated by including an immobilized
pepsin column to the LC setup before the peptide trapping
column for desalting. The high concentration of guanidine
HCl introduced in the H/D quenching step would be
detrimental to the pepsin activity. Therefore, after quenching,
five-fold dilution of LDH sample into 0.1% formic acid (pH
2.5) was performed. The peptide mixture resulting from
pepsin column digestion was desalted with the peptide
trapping column described above, and then separated in a
second peptide-resolving column (Kinetex 2.6 μmC18, 2.10×
100 mm, Phenomenex, Torrance, CA). For good peptide
separation, a shallower ACN elution gradient (from 15 to
40% over 20 min) was used. All the reporter peptides used
for HX analysis were assigned by performing tandem (MS/
MS) mass spectrometry, followed by analysis with TurboSEQUEST
software. To minimize artifactual isotope exchange
during the analysis time, all the columns, loops, and lines
were immersed in an ice bath during all the experiments.
The deuteration level for intact molecule and each
reporter peptide was calculated by the following equation:
D% ¼ m m0
ðm100 m0Þ
100% ð1Þ
where m is the measured centroid mass of the deuterated
molecule or peptide after a particular labeling time, and m0
and m100 are the two centroid mass limits of a molecule and
reporter peptide from zero-deuteration and full-deuteration
control experiments, respectively.
setup was used for mass spectrometry analysis. For whole
molecule HX analysis, after quenching of H/D exchange
with 50 mM citric buffer (pH 2.5, containing 6 M
guanidine HCl), LDH sample was immediately loaded into
a sample loop. An isocratic pump delivered the LDH
sample in the loop to a peptide trapping column (1 mm
ID×8 mm, catalog No. TR1/25108/01; Michrom Bioresources,
Auburn, CA) for desalting. After 6 min desalting,
a short gradient of acetonitrile (ACN) from 25% to 90%
over 7 min (50 μL/min by Surveyor MS Pump, Thermo,
San Jose, CA) was used to elute LDH from the trapping
column and deliver it to the electrospray ionization ion-trap
mass spectrometer (LTQ, Thermo Electron Corporation,
San Jose, CA).
In the peptide level HX analysis, an in-line proteolytic
digestion was incorporated by including an immobilized
pepsin column to the LC setup before the peptide trapping
column for desalting. The high concentration of guanidine
HCl introduced in the H/D quenching step would be
detrimental to the pepsin activity. Therefore, after quenching,
five-fold dilution of LDH sample into 0.1% formic acid (pH
2.5) was performed. The peptide mixture resulting from
pepsin column digestion was desalted with the peptide
trapping column described above, and then separated in a
second peptide-resolving column (Kinetex 2.6 μmC18, 2.10×
100 mm, Phenomenex, Torrance, CA). For good peptide
separation, a shallower ACN elution gradient (from 15 to
40% over 20 min) was used. All the reporter peptides used
for HX analysis were assigned by performing tandem (MS/
MS) mass spectrometry, followed by analysis with TurboSEQUEST
software. To minimize artifactual isotope exchange
during the analysis time, all the columns, loops, and lines
were immersed in an ice bath during all the experiments.
The deuteration level for intact molecule and each
reporter peptide was calculated by the following equation:
D% ¼ m m0
ðm100 m0Þ
100% ð1Þ
where m is the measured centroid mass of the deuterated
molecule or peptide after a particular labeling time, and m0
and m100 are the two centroid mass limits of a molecule and
reporter peptide from zero-deuteration and full-deuteration
control experiments, respectively.
HX Procedure to Detect Freezing-Induced LDH
Denaturation in the Frozen State
HX labeling in the frozen state was conducted according to
the protocol shown in Fig. 1A. LDH stock solution following
dialysis and centrifugation was first diluted to desired
protein concentrations in H2O-based citrate buffer
(20 mM sodium citrate, 100 mM NaCl, pH 6.2). The
diluted LDH sample was then mixed at 1:9 volume ratio
with D2O-based citrate buffer with the same solution
composition and pH. The resultant LDH samples had
90% deuterium in solvent and desired final protein
concentrations of 0.02, 0.05 and 0.10 mg/ml. To minimize
H/D exchange in the solution phase before sample
freezing, the final mixing step of LDH solution and
deuterium buffer was carried out in the shortest time
possible for manual operation (less than 2 s), immediately
followed by flash freezing in liquid N2 for 5 min. Frozen
LDH samples were then incubated for the selected labeling
time at −10°C. This temperature was chosen because H/D
exchange took place within a reasonable experimental time
scale (e.g. hours to one day). Carrying out the labeling at a
temperature lower than the eutectic point of NaCl solution
(−21.2°C) would completely freeze the sample (i.e. no
remaining liquid in the frozen sample), thus leading to an
extremely slow H/D exchange rate. Thawing of the
deuterium-labeled frozen sample was achieved by adding
twice the sample volume of ice-cold 50 mM citrate buffer
(pH 2.5) containing 6 M guanidine HCl. Consequently,
back exchange was minimized by lowering the pH to ~2.8
during the thawing period. Further, the time for complete
thawing of the frozen sample was reduced to 2 min by the
high concentration of guanidine HCl.
HX labeling in the frozen state was conducted according to
the protocol shown in Fig. 1A. LDH stock solution following
dialysis and centrifugation was first diluted to desired
protein concentrations in H2O-based citrate buffer
(20 mM sodium citrate, 100 mM NaCl, pH 6.2). The
diluted LDH sample was then mixed at 1:9 volume ratio
with D2O-based citrate buffer with the same solution
composition and pH. The resultant LDH samples had
90% deuterium in solvent and desired final protein
concentrations of 0.02, 0.05 and 0.10 mg/ml. To minimize
H/D exchange in the solution phase before sample
freezing, the final mixing step of LDH solution and
deuterium buffer was carried out in the shortest time
possible for manual operation (less than 2 s), immediately
followed by flash freezing in liquid N2 for 5 min. Frozen
LDH samples were then incubated for the selected labeling
time at −10°C. This temperature was chosen because H/D
exchange took place within a reasonable experimental time
scale (e.g. hours to one day). Carrying out the labeling at a
temperature lower than the eutectic point of NaCl solution
(−21.2°C) would completely freeze the sample (i.e. no
remaining liquid in the frozen sample), thus leading to an
extremely slow H/D exchange rate. Thawing of the
deuterium-labeled frozen sample was achieved by adding
twice the sample volume of ice-cold 50 mM citrate buffer
(pH 2.5) containing 6 M guanidine HCl. Consequently,
back exchange was minimized by lowering the pH to ~2.8
during the thawing period. Further, the time for complete
thawing of the frozen sample was reduced to 2 min by the
high concentration of guanidine HCl.
ANS and ThT Binding Fluorescence Analysis
Fluorescence analysis of ANS and ThT binding to F/Tinduced
aggregated sample was performed at room temperature
on a FluoroMax-3 spectrofluorometer (Horiba Jobin
Yvon, Edison, NJ). A quartz cell with 5 mm path length (Part
number 4ES5X5 Precision Cells Inc., Farmingdale, NY) was
used. For ANS binding analysis, 360 μL LDH samples
(0.1 mg/ml) were mixed with 40 μL of 100 μM ANS stock
solution. Mixed samples were excited at 390 nm, and the
emission spectrum was recorded over a wavelength range of
410–600 nm. For ThT binding analysis, 360 μL 0.1 mg/ml
LDH samples were mixed with 40 μL of 100 μM ThT stock
solution. The excitation wavelength was 446 nm, and the
emission spectrum was recorded over a wavelength range of
460–560 nm. All the measurements were performed within
5 min of sample mixing for both ANS and ThT binding, and
three scans were performed to obtain an average spectrum for
each sample.
aggregated sample was performed at room temperature
on a FluoroMax-3 spectrofluorometer (Horiba Jobin
Yvon, Edison, NJ). A quartz cell with 5 mm path length (Part
number 4ES5X5 Precision Cells Inc., Farmingdale, NY) was
used. For ANS binding analysis, 360 μL LDH samples
(0.1 mg/ml) were mixed with 40 μL of 100 μM ANS stock
solution. Mixed samples were excited at 390 nm, and the
emission spectrum was recorded over a wavelength range of
410–600 nm. For ThT binding analysis, 360 μL 0.1 mg/ml
LDH samples were mixed with 40 μL of 100 μM ThT stock
solution. The excitation wavelength was 446 nm, and the
emission spectrum was recorded over a wavelength range of
460–560 nm. All the measurements were performed within
5 min of sample mixing for both ANS and ThT binding, and
three scans were performed to obtain an average spectrum for
each sample.
Dynamic Light Scattering (DLS)
LDH aggregate size distribution was measured by dynamic
light scattering at 25°C with a DynaPro Plate Reader
(Wyatt Technology, Santa Barbara, CA). All LDH samples
were diluted to 0.1 mg/ml before measurement. Scattered
light intensity was collected at 90º to the incident light. The
signal intensity was then averaged over a five-second period
to obtain autocorrelation functions using manufacturer’s
software, DYNAMICS (Wyatt Technology, Santa Barbara,
CA). Twenty acquisitions were averaged to make one
measurement, and three measurements were conducted for
each LDHsample. In data analysis, aggregate size distribution
and polydispersity were determined using the regularization
method implemented in the DYNAMICS software.
light scattering at 25°C with a DynaPro Plate Reader
(Wyatt Technology, Santa Barbara, CA). All LDH samples
were diluted to 0.1 mg/ml before measurement. Scattered
light intensity was collected at 90º to the incident light. The
signal intensity was then averaged over a five-second period
to obtain autocorrelation functions using manufacturer’s
software, DYNAMICS (Wyatt Technology, Santa Barbara,
CA). Twenty acquisitions were averaged to make one
measurement, and three measurements were conducted for
each LDHsample. In data analysis, aggregate size distribution
and polydispersity were determined using the regularization
method implemented in the DYNAMICS software.
High Performance Size Exclusion Chromatography (HPSEC)
HPSEC was performed on a BioLogic DuoFlow chromatography
system (BioRad, Hercules, CA). LDH samples (150 μL,
sample concentration), either native or aggregated, after a
number of F/T cycles were loaded onto a prequilibrated
TSKgel SEC column (G3000 SWXL, 7.8 mm×30 cm,
Tosoh Bioscience, Bellefonte, PA), and then eluted with 0.3M
NaCl, 50 mM PBS (pH 7.0). The elution flow rate was 1 ml/
min. LDH aggregation was expressed as the loss of monomer
peak after normalization to the peak area of a native LDH
sample. Three replicates were performed for each LDH
sample to determine the aggregation level after repeated F/T
cycles and the associated standard deviation.
system (BioRad, Hercules, CA). LDH samples (150 μL,
sample concentration), either native or aggregated, after a
number of F/T cycles were loaded onto a prequilibrated
TSKgel SEC column (G3000 SWXL, 7.8 mm×30 cm,
Tosoh Bioscience, Bellefonte, PA), and then eluted with 0.3M
NaCl, 50 mM PBS (pH 7.0). The elution flow rate was 1 ml/
min. LDH aggregation was expressed as the loss of monomer
peak after normalization to the peak area of a native LDH
sample. Three replicates were performed for each LDH
sample to determine the aggregation level after repeated F/T
cycles and the associated standard deviation.
Rabu, 14 Desember 2011
Ammonia Poisoning
Ammonia is a toxic gas if it melepasi Had a certain density. For workers who are exposed to ammonia, which is justified must necessarily exposure below 25 ppm (ACGIH). More than that, things should not be accepted namely merbahaya. Ammonia Odor threshold also has a low (5 ppm). So, if the smell of ammonia stench was first dikesan, things were still permitted to ansur rejected by the human body. If you make a visit refinery products such as latex berasaskan cathetter, tiub and so on, you will be able to see the workers who sentiasa sniff the smell of Ammonia. This situation will probably lead to workers is less sensitive to the dangers of Ammonia. Someone who is familiar with the smell will not squeeze something if kepekatannya increase. This may jeopardize kerana they probably will not comply with safety measures in the workplace.
In KES prevailing in Tg Karang is also, by kerana Ammonia pure / almost pure persekitaran ekoran released into the leak, then the concentration of ammonia in the air in persekitaran almost to where the leak was typically very high, and far exceeding the STEL (Short term exposure limit) or MEL (maximum exposure limit). This causes shortness of breath when they menghidunya.
This event clearly shows the importance of safety consciousness use of chemicals in the workplace as outlined in the regulations of safety and safety in the Deed kesihataan job (OSHA), 1994, his trademark regulations Use and Standards of Exposure Chemical Hazardous to Health, issued in the year USECHH 2000.
As a first step, workers need to be identified the hazards that form in their persekitaran. You may get a notice about the dangers of something in the chemical material safety data sheet (CSDS), which is also recognized as a material safety data sheets (MSDS). Today's announcement is very easily found. With only menaip MSDS (material name) on search engines like google, you will be able to find edict fizikokimia and toxicological properties sentiasa present in all MSDS. If you are an employer, you need to explain the dangers to workers kerana it is the responsibility of you, If you're working, you need to know your own danger kerana him to be exposed.
Reference : http://arshadahmad.wordpress.com/2009/08/12/5-maut-akibat-keracunan-ammonia/
In KES prevailing in Tg Karang is also, by kerana Ammonia pure / almost pure persekitaran ekoran released into the leak, then the concentration of ammonia in the air in persekitaran almost to where the leak was typically very high, and far exceeding the STEL (Short term exposure limit) or MEL (maximum exposure limit). This causes shortness of breath when they menghidunya.
This event clearly shows the importance of safety consciousness use of chemicals in the workplace as outlined in the regulations of safety and safety in the Deed kesihataan job (OSHA), 1994, his trademark regulations Use and Standards of Exposure Chemical Hazardous to Health, issued in the year USECHH 2000.
As a first step, workers need to be identified the hazards that form in their persekitaran. You may get a notice about the dangers of something in the chemical material safety data sheet (CSDS), which is also recognized as a material safety data sheets (MSDS). Today's announcement is very easily found. With only menaip MSDS (material name) on search engines like google, you will be able to find edict fizikokimia and toxicological properties sentiasa present in all MSDS. If you are an employer, you need to explain the dangers to workers kerana it is the responsibility of you, If you're working, you need to know your own danger kerana him to be exposed.
Reference : http://arshadahmad.wordpress.com/2009/08/12/5-maut-akibat-keracunan-ammonia/
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