Chem 453 Quiz 1 !!! Do in Pairs!
NAMES
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Attach requested data to this sheet.
1) We will soon be purifying and analyzing a protein called Alkaline Phosphatase (AP) from E.
coli. Go to http://ca.expasy.org/ and find and copy the amino acid sequence of the HUMAN
PLACENTAL version of this protein (or its precursor with signal).
>sp|P05187|PPB1_HUMAN Alkaline phosphatase, placental type
precursor (EC 3.1.3.1) (PLAP-1) (Regan isozyme) - Homo sapiens
(Human).
MLGPCMLLLLLLLGLRLQLSLGIIPVEEENPDFWNREAAEALGAAKKLQPAQTAAKNLII
FLGDGMGVSTVTAARILKGQKKDKLGPEIPLAMDRFPYVALSKTYNVDKHVPDSGATATA
YLCGVKGNFQTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASPAG
TYAHTVNRNWYSDADVPASARQEGCQDIATQLISNMDIDVILGGGRKYMFRMGTPDPEYP
DDYSQGGTRLDGKNLVQEWLAKRQGARYVWNRTELMQASLDPSVTHLMGLFEPGDMKYEI
HRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGHHESRAYRALTETIMFDDAI
ERAGQLTSEEDTLSLVTADHSHVFSFGGYPLRGSSIFGLAPGKARDRKAYTVLLYGNGPG
YVLKDGARPDVTESESGSPEYRQQSAVPLDEETHAGEDVAVFARGPQAHLVHGVQEQTFI
AHVMAFAACLEPYTACDLAPPAGTTDAAHPGRSVVPALLPLLAGTLLLLETATAP
2) Go to Prot-Param database (http://ca.expasy.org/tools/protparam.html) and find the pI
(isoelectric point) of AP (without precursor signal sequence). What charge would this protein
have at pH 7.0?
Theoretical pI: 5.74
Negative
3) What is the extinction coefficient estimate of AP at 280 nm and calculate the following.
Assume no half-cysteines:
 =47330
You have purified AP and have 76.0 mL of a solution giving an absorbance of 0.87 at 280 nm in
a 1 cm cuvette. How many MOLES of AP do you have? How many GRAMS of AP do you have?
0.87=47330*1*c c= 1.8 X 10-5 mol/L
1.8 X 10-5 mol/L* .076 L= 1.4 X 10-6 moles
1.4 X 10-6 moles X 52745.3 g/mol = 0.074 g or 74 mg