Calculations: Determination for g of protein Using Table 1 and tube 2: Concentration of standard protein solution = 200g/mL Volume of standard

solution in tube 2 = 0.1mL 1mL of standard protein solution = 200g 0.1mL of standard protein solution = (200 x 0.1)/1 = 20g

Average Absorbance Using tubes 4&5 of Table 1: = (0.283+0.281)/2 = 0.282

Determination of Protein Content in Diluted Fractions The absorbance reading (X100 dilution) for Fraction 1 (Homogenate) at 750nm was 0.502. Using the equation of the calibration curve: y = 0.0026x 0.502 = 0.0026x x = 0.502/0.0026 = 193.08 Therefore in Fraction 1, X100 dilution, there was 193.08g of protein as seen in Table 2.

Determination of Protein Concentration in X100 dilution (mg/mL) In X100 dilution of Fraction 1 there is 193.08g of protein Therefore, 1mL of undiluted fraction contains 193.08 x 100 = 19308g protein Since 1000g = 1mg 19308g = (1/1000) x 19308

= 19.31mg

Determination of Protein Concentration in X200 dilution (mg/mL) In X200 dilution of Fraction 1 there is 138.46g of protein Therefore, 1mL of undiluted fraction contains 138.46 x 200 = 27692g protein Since 1000g = 1mg 27692g = (1/1000) x 27692 = 27.69mg

Determination of Average = (27.69 + 19.31)/2 = 23.5mg/mL

Determination of Total Protein (mg)= Average x Total volume of Homogenate (Fraction 1) = 23.5mg/mL x 760mL = 17860mg

Determination of % Recovery= ((Total protein of mitochondrial fraction/ Total protein of homogenate (F1) )x 100 = (17860/17860) x 100 = 100%

Determination for mol of phosphorous Concentrated stock KH2PO4= 2.18mg/100mL 136mg KH2PO4 = 1mmol 2.18mg KH2PO4 = (1/136) x 2.18 = 0.016mmol = 16mol Using tube 2 of Table 4,

100mL of phosphate solution = 16mol 0.5mL of phosphate solution = (16/100) x 0.5 = 0.08mol KH2PO4

Determination for g of phosphorous Molar Ratio 1:1 KH2PO4 : P 1mol KH2PO4 = 30.97g P Using tube 2 of Table 4, 0.08mol KH2PO4 = (30.97 x 0.08)/1 = 2.48g of P

Determination of Phosphate Content (mol) in Fractions The absorbance reading for Fraction 1 (Homogenate) at 650nm was 0.105. Using the equation of the calibration curve: y = 1.1232x 0.105 = 1.1232x x = 0.105/1.1232= 0.093 Therefore in Fraction 1, there was 0.093 of phosphate as seen in Table 6.

Determination of Activity for Glucose-6-Phosphatase Using Table 7, Fraction 1: 0.093mol of P from 1mL of supernatant, This was taken from 2mL of solution therefore: 2mL of solution contained: 0.093 x 2 = 0.186 mol of P This 2mL solution contained 0.1mL of diluted sample therefore: 0.1mL of diluted sample contains 0.186 mol of P; 1mL of diluted sample would contain: (0.186/0.1)x1 = 1.86mol of P

This reaction occurred in 10 mins hence: In 1 minute 1mL of sample would contain: (1.86/10) x1 = 0.186 mol of P/min/mL; This was obtained from sample diluted 10x therefore: 1mL of undiluted sample would have: 0.186 x 10 = 1.86 mol of P/min/mL; Since 1 mol of P/min = U Undiluted Sample activity = 1.86 U/mL Specific activity: From Table 3: 1mL of sample contained 23.50 mg of protein Therefore in 23.50 mg of protein there was 1.86 U of activity; 1mg would contain: (1.86/23.5) x 1 = 0.079 U/mg Total Activity: Total volume of Fraction (mL) x Activity (U/mL); 760 x 1.86 = 1413.6 U of activity. % Total Activity: (Total activity of F1/Sum of Total Activity of F2,F3,F4,F5) x 100; (1413.6/1399.1) x 100 = 101%

Determination for mol of p-nitrophenol: 1000mL contains 100mol of p-nitrophenol; 0.2mL would contain: (100/1000) x 0.2 = 0.02mol of p-nitrophenol

Determination of p-nitrophenol Content (mol) in Fractions The absorbance reading for Fraction 1 (Homogenate) at 405nm was 0.440. Using the equation of the calibration curve: y = 0.4558x 0.440 = 0.4558x

x = 0.440/0.4558= 0.97 Therefore in Fraction 1, there was 0.97 of p-nitrophenol as seen in Table 9.

Determination of Activity of Acid Phosphatase Assay: Using Fraction 1 of Table 10, 0.97 mol of p-Nitrophenol was present in 4 mL of solution; This solution contained 0.2mL of diluted sample hence; 0.2mL of diluted sample reacts to form: 0.97mol of p-Nitrophenol; In 1mL of diluted sample would contain: (0.97/0.2) x 1 = 4.85mol of p-Nitrophenol; This reaction took place over 10 minutes, therefore: In 1 minute: (4.85/10) x 1 = 0.485 mol of p-Nitrophenol/min This was taken from a 1 in 10 sample hence; In 1mL of undiluted sample would react to form: 0.485 x 10 = 4.85 mol of pNitrophenol/min/mL = 4.85 U/mL

Specific Activity: 1mL of undiluted homogenate contains: 23.5mg of protein/mL If 1mL has an activity of 4.85U then 23.5mg of protein has 4.85U of activity; 1mg would have: (4.85/23.5) x 1 = 0.21U/mg Total Activity: 1mL of homogenate contains: 4.85U/mL; In 760mL of homogenate would have: 4.85 x 760 = 3686U % Total Activity: (3686/2403) x 100 = 153.4%

Latent Activity (lysosomes): Total activity (Triton X) free activity (no Triton X) = 66-240= -174

Change in Absorbance/min for Glutamate Dehydrogenase in homogenate: Average Abs at 30 sec = (0.713+0.655)/2 = 0.684 Average Abs at 240 sec = (0.650+0.607)/2 = 0.629 Change in Abs/min = Abs @ 30 sec Abs @ 240 sec/ 4 = (0.684-0.629)/4 = 0.014

Lactate Dehydrogenase in homogenate: Average Abs at 30 sec = (0.522+0.548)/2 = 0.535 Average Abs at 240 sec = (0.151+0.156)/2 = 0.154 Change in Abs/min = Abs @ 30 sec Abs @ 240 sec/ 4 = (0.535-0.154)/4 = 0.095

Determination for Activity of Glutamate Dehydrogenase: For Homogenate: From equation A = C x el where: C refers to concentration in moles, A refers to Absorbance, el refers to the molar extinction coefficient of NADH (6220 cm-1 M-1 ). Converted to: C = A/el; 0.014/6220 = 2.25 x 10-6

To moles: Since 1mol= 1000000mol 2.25 x 10-6mol= (1000000/1) x 2.25 x 106 = 2.25 moles/min/litre

To moles/min/mL: 2.25 /1000 = 2.25 x 10-3moles/min/mL This was in a 1 in 50 diluted solution hence: In undiluted sample: 2.25x 10-3x 50 = 0.113 moles/min/mL = 0.113U/mL Specific Activity: In 1mL of homogenate contains 23.5mg of protein. Therefore the activity in 1g of protein = 0.113 / 23.5 = 0.0048 U/mg or 4.8 x10-3 U/mg

Determination of mol of NT: 0.05%NT 0.05g in 100ml Now 662g = 1mol NT 0.05g = (1/662) x 0.05 = 7.6 x 10-5 mol NT = 76mol NT 100ml = 76mol NT Therefore, using tube 2 of Table 16, 0.1ml = (76/100) x 0.1 = 0.076mol NT

Determination of NT Content (mol) in Fractions The absorbance reading for Fraction 1 (Homogenate) at 540nm was 0.040. Using the equation of the calibration curve: y = 1.0592x 0.040 = 1.0592x x = 0.040/1.0592= 0.038

Therefore in Fraction 1, there was 0.038mol of NT as seen in Table 17.

Determination of Activity for Succinate Dehydrogenase: Using Fraction 1 of Table 18, 0.3mL of diluted sample = 0.038 mol of NT The ratio of Succinate : NT is 2:1 so 0.038 x 2 = 0.076molNT As such, 1mL of diluted sample = (0.076/0.3) x 1 = 0.253molNT This reaction took place over 20 minutes, therefore: In 1 minute: (0.253/20) x 1 = 0.013 mol of NT/min This sample was not diluted therefore: 1mL of undiluted sample = 0.013mol of NT/min/mL; Since 1 mol of P/min = U Undiluted Sample activity = 0.013 U/mL

Specific Activity: 1mL of undiluted homogenate contains: 23.5mg of protein/mL If 1mL has an activity of 0.013U then 23.5mg of protein has 0.013U of activity; 1mg would have: (0.013/23.5) x 1 = 5.53 x 10-4U/mg Total Activity: 1mL of homogenate contains: 0.013U/mL; In 760mL of homogenate would have: 0.013 x 760 = 9.88U