Sample # Vol 0.1 M Buffer pH=10 /uL Vol 0.

Vol 0.3m M PNP /uL Vol Total /uL [PNP]/mM Sample # [PNP]/mM A405
1 1000 0 1000 0 1 0 0 PNP Standard Curve
2 900 100 1000 0.03 2 0.03 0.56635 4
3 800 200 1000 0.06 3 0.06 1.13035
3.5 f(x) = 18.807487745098 x + 0.00131899509803946
4 700 300 1000 0.09 4 0.09 1.69435 R² = 0.999999699311229
5 600 400 1000 0.12 5 0.12 2.25835 3

6 500 500 1000 0.15 6 0.15 2.82235 2.5

7 334 666 1000 0.2 7 0.2 3.76235

A405
2
1.5
l=1 cm e0 = Slope/l = 18.807/1 1/(mM.cm) 1
0.5
e0l = slope 18.807 1/mM 0
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2
[PNP]/mM
Sample # Vol DW /uL Vol 0.1 M Buffer pH=10 /uL Vol 1m M PNPP /uL Vol 50 ug/mL ALP /uL Vol Total /uL [ALP]/(ug/mL) [PNPP]/uM
1 800 100 0 100 1000 5 0
2 790 100 10 100 1000 5 10
3 780 100 20 100 1000 5 20
4 760 100 40 100 1000 5 40
5 740 100 60 100 1000 5 60
6 720 100 80 100 1000 5 80
7 700 100 100 100 1000 5 100
8 600 100 200 100 1000 5 200
 Sample #
1 2 3 4 5 6 7 8 Sample # Slope of tangent at zero D [PNP]/D t ( mM/min)
time/min A405 [PNP]/mM time/min A405 [PNP]/mM time/min A405 [PNP]/mM time/min A405 [PNP]/mM time/min A405 [PNP]/mM time/min A405 [PNP]/mM time/min A405 [PNP]/mM time/min A405 [PNP]/mM 1 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0.491 0.491
1 0 0 1 0.980 0.184 1 1.225 0.230 1 1.470 0.276 1 1.715 0.322 1 1.960 0.368 1 2.205 0.415 1 2.450 0.461 3 0.555 0.555
2 0 0 2 0.996 0.187 2 1.245 0.234 2 1.494 0.281 2 1.743 0.328 2 1.992 0.375 2 2.241 0.421 2 2.490 0.468 4 0.593 0.593
3 0 0 3 1.006 0.189 3 1.257 0.236 3 1.508 0.284 3 1.760 0.331 3 2.011 0.378 3 2.263 0.425 3 2.514 0.473 5 0.608 0.608
4 0 0 4 1.012 0.190 4 1.265 0.238 4 1.519 0.285 4 1.772 0.333 4 2.025 0.381 4 2.278 0.428 4 2.531 0.476 6 0.615 0.615
5 0 0 5 1.018 0.191 5 1.272 0.239 5 1.527 0.287 5 1.781 0.335 5 2.035 0.383 5 2.290 0.430 5 2.544 0.478 7 0.619 0.619
6 0 0 6 1.022 0.192 6 1.278 0.240 6 1.533 0.288 6 1.789 0.336 6 2.044 0.384 6 2.300 0.432 6 2.555 0.480 8 0.629 0.629
7 0 0 7 1.026 0.193 7 1.282 0.241 7 1.539 0.289 7 1.795 0.337 7 2.052 0.386 7 2.308 0.434 7 2.564 0.482
8 0 0 8 1.029 0.193 8 1.286 0.242 8 1.543 0.290 8 1.801 0.339 8 2.058 0.387 8 2.315 0.435 8 2.572 0.484
1
9 0 0 9 1.032 0.194 9 1.290 0.242 9 1.548 0.291 9 1.806 0.339 9 2.064 0.388 9 2.322 0.436 9 2.580 0.485 [ PNP ]  A 405
10 0 0 10 1.034 0.194 10 1.293 0.243 10 1.552 0.292 10 1.810 0.340 10 2.069 0.389 10 2.327 0.438 10 2.586 0.486
 0l
Sample 2 Sample 4 Sample 6 Sample 8
0.196 0.295
Y [PNP ]
0.395 0.490
0.194 0.485
0.290 0.390
slope  
0.192 f(x) = − 0.00011827609006978 x² + 0.00232798952426587 x f(x) = − 0.00017741413510468 x² + 0.00349198428639883 x f(x) = − 0.00029569022517446 x² + 0.00581997381066467 x
+ 0.182660388879915 0.385 f(x) = − 0.00023655218013957 x² + 0.00465597904853174 x 0.480
0.190 0.285 + 0.273990583319873 + 0.456650972199789
0.475
X t
R² = 0.987432917642845 + 0.365320777759831
[PNP]/mM

R² = 0.987432917642846 R² = 0.987432917642847

[PNP]/mM
0.380

[PNP]/mM
[PNP]/mM
0.188 R² = 0.987432917642845 0.470
0.280 0.375
0.186 0.465
0.184 0.275 0.370 0.460
0.182 0.365 0.455
0.270
0.180 0.360 0.450 dy dy
0.178
0 1 2 3 4 5 6 7 8 9 10
0.265
0 1 2 3 4 5 6 7 8 9 10 0.355
0 1 2 3 4 5 6 7 8 9 10
0.445
0 1 2 3 4 5 6 7 8 9 10
slope of tan gent  
dx
aX 2  bX  c  b
time/min time/min time/min time/min dx 0

Sample 3 Sample 5 Sample 7 Sample 2+3

0.245 0.345 0.440 0.250
0.340 0.435 f(x) = − 0.00026612120265702 x² + 0.00523797642959829 x 0.240
0.240 f(x) = − 0.00014784511258723 x² + 0.00290998690533233 x f(x) = − 0.00014784511258723 x² + 0.00290998690533233 x
+ 0.228325486099894 f(x) = − 0.00020698315762212 x² + 0.00407398166746525 x 0.430 + 0.41098587497981 0.230 + 0.228325486099894
0.335 + 0.319655680539852 R² = 0.987432917642847 0.220
R² = 0.987432917642847 0.425 R² = 0.987432917642847

[PNP]/mM
[PNP]/mM

[PNP]/mM

0.235

[PNP]/mM
0.330 R² = 0.987432917642847 0.210
0.420 0.200
0.230 0.325 0.415 0.190 f(x) = − 0.00011827609006978 x² + 0.00232798952426587 x
0.320 0.410 0.180 + 0.182660388879915
0.225 0.170 R² = 0.987432917642845
0.315 0.405
0.160
0.220 0.310 0.400 0.150
0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10
time/min time/min time/min time/min
Sample # [PNPP]/uM D [PNP]/D t ( mM/min) n0 /(umol/min) uM/[PNPP] (umol/min)/n0
1 0 0 0 - - ALP-Activity Lineweaver-Burk Plot
2 10 0.491 0.491 0.100 2.038 0.650 1.850
3 20 0.555 0.555 0.050 1.803
1.800
4 40 0.593 0.593 0.025 1.685 0.600 f(x) = 4.709831 x + 1.567321
5 60 0.608 0.608 0.017 1.646 1.750 R² = 1

n0 /(umol/min)

(umol/min)/n0
6 80 0.615 0.615 0.013 1.626 0.550 1.700
7 100 0.619 0.619 0.010 1.614 1.650
8 200 0.629 0.629 0.005 1.591 0.500 1.600
1.550
ALP 0.450
PNPP PNP + Pi 1.500
H2O 0.400 1.450
0 20 40 60 80 100 120 140 160 180 200 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 0.040 0.045 0.050

1  [ PNP ]  1  1   A 405  [PNPP]/uM uM/[PNPP]

0   V T 0    l   t V T
 t   0  
KM = 3.005
max [ PNPP ] 1 1 K  1
0    M
K M  [ PNPP ] 
 1 1  1   A 405 
0  
0 max  max  [PNPP ]
n max = 0.638 (umol/min)
 V T
   0 l   t 
V T 1mL
 KM   slope 
slope   K M  
  max   Y  Int 
1 1
Y  Int  max 
 max Y  Int