detector
1. Origin
ŝ
2
ŝ o
  

Define so  k o , s  k ,  = scattering angle
Scattering depends on environment and angle,
Angle depends on s and so.
 ˆs ˆs o
s   , [s] = cycles/cm , scattering vector
 
1
 ˆs 2  ˆs 2  2ˆs  ˆs  2
o
o
1/2
.
|s| = (s s) = 


2



1/2
= (1/(2 – 2 cos(2)) = (1/(2 (1 - cos(2))1/2
= (1/(4sin2())1/2 = (2/|sin(
So,
0 < |s| < 2/reciprical space.
1
Cos(2) = cos2() - sin2(), 1 – cos(2) = 2sin2()
2. From electron at r.
ŝ
ŝ o
x1
1
2
ŝ
r
2
x2
ŝ o
Path length difference is x2 – x1
x
x
 r 2 r 1
r
r
 rcos 2   rcos1 


 r  sˆ   r  sˆo 
So,


 
ˆs  so   r

sr
=

s11 kinetics HbS
3500000
3000000
2000000
s11
1500000
1000000
500000
0
0
500
1000
1500
2000
time
Effect of GSNO on HbS polymerization
in 1.8M phosphate buffer, pH 7.4 (Trial1)
2
Absorption (700nm)
s11
2500000
1.5
0mM
0.25mM
1
1.25mM
2.5mM
25mM
0.5
0
0
10
20
Time (min)
30
40
Figure 4. Time delay measurement for 100% tetranitrosyl HbS compared to
deoxy HbS. The turbidity was observed using the Cary 100 Bio UV visible
spectrometer at 800 nm using 0.1 cm path length cells. The concentrations of
the 100% tetranitrosyl and deoxy samples were 16 mM. The delay times were
calculated as the time to reach one-tenth of the final optical density after
subtraction of the initial absorption at time t = 0. The delay time for the deoxy
sample was 110 sec while the 100% tetranitrosyl sample was greater than 7200
sec (possibly infinity).
2.5
(a)
Extinction
2
0°C, 0th min
1.5
15°C, 90th min
1
37°C, 30th min
37°C, 38th min
0.5
0
450
37°C, 50th min
500
550
600
650
700
Wavelength (nm)
Figure 5. Dexoygenated HbS samples contained in square cuvettes. (a)
Extinction (absorbance + turbidity) of deoxygenated HbS samples (0.09 mM) in
1.8M phosphate. Samples were temperature jumped from 0 oC () to 15 oC ()
or 37 oC and extinction was measured at various times (30 minutes (), 38
minutes (), and 50 minutes () after being jumped to 3 7oC). The extinction at 0
oC and 15 oC were exactly same (and overlap each other in the figure), indicating
that no HbS polymers formed at 15 oC.
(a)
I * Sin( ) / C
1.60
1.20
HbA/PBS
HbA/1.5Mphosphate
HbS/1.5Mphosphate
0.80
HbS/1.8Mphosphate
HbS/Dextran * 7
0.40
0.00
25
60
95
130
Angle (degrees)
-6
Kc/R (x10 )
16
(b)
12
HbA/PBS
HbA/1.5Mphos
HbS/1.5MPhos
HbS/1.8MPhos
8
4
0
0
0.5
1
sin2( /2)
Figure 1. Light scattering from Hb in different buffers. (a) Static light
scattering of HbA and HbS as a function of scattering angle. The
concentrations of HbA in PBS (), HbA in 1.5 M phosphate (), HbS in 1.5 M
phosphate (), HbS in 1.8 M phosphate () and HbS in dextran (intensity
multiplied sevenfold, ) were 0.15 mM, 0.129 mM, 0.158 mM, 0.133 mM and
1.174 mM (heme), respectively. Scattering intensities were normalized by
protein concentrations. (b) Zimm plots of the static light scattering from
hemoglobin in different phosphate buffers in Figure 1(a). Zimm plots show
that the molecular weights of hemoglobin in high phosphate are large, but
the one of HbA in PBS is in the expected range. The average apparent
weight-average molecular weights of hemoglobin in different buffers are
summarized in Table 1.
Table 1. SLS apparent average molecular weight and radius of gyration of
Hemoglobin in different phosphate buffers
Hb in different Buffers
HbA in PBS
HbA in 1.5M phosphate
HbA in 1.8M phosphate
HbS in PBS
HbS in 1.5M phosphate
HbS in 1.8M phosphate
Mw(KDa)
694
71056
143071
704
125082
2200109
Rg(nm)
--21012
23915
--23011
2489