Question

Experiments A and B are 2 experiments performed by mixing alkaline phosphatase enzyme with Pnpp substrate. The reaction was run for 5 minutes for each tube, then stopped by adding NaOH.

A)Changing enzyme concentration

Tube	Reaction Buffer (mL)	5.4 mM of pNPP substrate (mL)	0.002 mg/ml of AP enzyme (mL)	3M of NaOH (mL)	Absorbance	Final Enzyme concentration (mM)	Velocity (umol/min/mL)
1	3.41	0.04	0.05	0.875	0.284
2	3.36	0.04	0.1	0.875	0.387
3	3.31	0.04	0.15	0.875	0.509
4	3.26	0.04	0.2	0.875	0.538
5	3.21	0.04	0.25	0.875	0.569
6	3.16	0.04	0.3	0.875	0.602
7	3.11	0.04	0.35	0.875	0.620
8	3.06	0.04	0.4	0.875	0.638

Calculations for each tube:

1)Calculate final enzyme concentrations in each tube

2)Use beers law to convert absorbance to concentrations (umol). Molar extinction coefficient of p-nitrophenol is 16.2 mM^-1cm^-1. Path length is 1 cm.

3)Calculate velocity (umol/min/mL), make sure to correct the concentration for NaOH addition

B)Changing ph

Tube	Reaction Buffer pH	Reaction Buffer (mL)	5.4 mM of pNPP substrate (mL)	0.002 mg/ml of AP enzyme (mL)	3M of NaOH (mL)	Absorbance	Final substrate concentration (mM)	Velocity (umol/min/mL)
1	7	3.39	0.04	0.07	0.875	0.194
2	7.5	3.39	0.04	0.07	0.875	0.244
3	8	3.39	0.04	0.07	0.875	0.276
4	8.5	3.39	0.04	0.07	0.875	0.347
5	9	3.39	0.04	0.07	0.875	0.451
6	10	3.39	0.04	0.07	0.875	0.292
7	11	3.39	0.04	0.07	0.875	0.102
8	12	3.39	0.04	0.07	0.875	0.056

Calculations for each tube:

1)Calculate final substrate concentrations

2)Use beers law to convert absorbance to concentrations (umol). Molar extinction coefficient of p-nitrophenol is 16.2 mM^-1cm^-1. Path length is 1 cm.

3)Calculate velocity (umol/min/mL), make sure to correct the concentration for NaOH addition

Answer 1

A)

1) The final Concentration of enzyme is the same to initial concentration of enzyme in each test tube. Because enzyme is catalyst and it regenerate in Evey cycle.

2)

Tube	Buffer	5.4 mM Pnpp Con		0.002 mg/ml AP	NaOH	Abs	Total Vol	Final enzyme con		Phenol con M		Velocity umol/min/ml
1	3.41	0.04		0.05	0.875	0.284	4.375	0.05		1.75309E-05		0.003506173
2	3.36	0.04		0.1	0.875	0.387	4.375	0.1		2.38889E-05		0.004777778
3	3.31	0.04		0.15	0.875	0.509	4.375	0.15		3.14198E-05		0.006283951
4	3.26	0.04		0.2	0.875	0.538	4.375	0.2		3.32099E-05		0.006641975
5	3.21	0.04		0.25	0.875	0.569	4.375	0.25		3.51235E-05		0.007024691
6	3.16	0.04		0.3	0.875	0.602	4.375	0.3		3.71605E-05		0.007432099
7	3.11	0.04		0.35	0.875	0.62	4.375	0.35		3.82716E-05		0.007654321
8	3.06	0.04		0.4	0.875	0.638	4.375	0.4		3.93827E-05		0.007876543

B) Changing pH

Tube		Buffer pH	buffer ml	5.4 mM Pnpp Con	AP ml	NaOH	Total Vol	ABS	Initial PnPP Con M	Phenol con M		Final Substrate con M		Velocity umol/min/ml
1		7	3.39	0.04	0.07	0.875	4.375	0.194	4.93714E-05	1.19753E-05		3.73961E-05		0.002395062
2		7.5	3.39	0.04	0.07	0.875	4.375	0.244	4.93714E-05	1.50617E-05		3.43097E-05		0.003012346
3		8	3.39	0.04	0.07	0.875	4.375	0.276	4.93714E-05	1.7037E-05		3.23344E-05		0.003407407
4		8.5	3.39	0.04	0.07	0.875	4.375	0.347	4.93714E-05	2.14198E-05		2.79517E-05		0.004283951
5		9	3.39	0.04	0.07	0.875	4.375	0.451	4.93714E-05	2.78395E-05		2.15319E-05		0.005567901
6		10	3.39	0.04	0.07	0.875	4.375	0.292	4.93714E-05	1.80247E-05		3.13467E-05		0.003604938
7		11	3.39	0.04	0.07	0.875	4.375	0.102	4.93714E-05	6.2963E-06		4.30751E-05		0.001259259
8		12	3.39	0.04	0.07	0.875	4.375	0.056	4.93714E-05	3.45679E-06		4.59146E-05		0.000691358