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11c Cat 3
Biochemistry 3723Lecture 11c Catabolite Inhibition #2Oct. 31, 2001
I.Measurement of sugars in medium--Day 3 of the experiment
A.Background--There are many colorimetric tests, both qualitative and quantitative, for sugars.
B.The tests we are using:
1.Enzymatic determination of glucose by glucose oxidase
a.Reaction--will detect free glucose only:
b.Coupled reaction to generate color
c.Use kit: Sigma Glucose Assay Kit--Dip sticks for monitoring blood or urine glucose use same type of rxn, but different chromophores.
2.Anthrone test for total carbohydrate
a.Reaction
i.Hydrolysis to monosaccharides (dimers through polymers react)
ii.Dehydration-- product is a furfural.
iii. reaction of furfural with anthrone
iv. Anthrone reacts non-specifically with all carbohydrates except alditols and amino sugars. Will react with lactose and glucose. Lactose gives ≈ twice the color intensity (why?).
b.Reduce sugars to allow lactose to be measured specifically in solutions containing both lactose and glucose
i. Reduction by NaBH4 of glucose makes sorbitol (an alditol) which does not react with anthrone.
ii. Reduction of lactose makes lactitol,
c.After BH4 reduction do anthrone test
i.sorbitol no reaction with anthrone
ii.lactitol hydrolyzes to sorbitol + galactose:
C.Practical comments
1.Glucose oxidase--a coupled assay (reaction goes to completion in 45 min, 25°C, dark).
a.Prepare standard curve with known glucose concentrations
b.Run lactose control--what does this tell you?
c.Chromogen: as any such compound this is potentially toxic. Disposal?
2.BH4 reduction
a.Competing reaction:BH4-+ H+ + 3 H2O ––> 4 H2 + H3BO3
H2 is explosive!
b.Precautions:
i.Work in hood, wear goggles
ii.Control pH during BH4 reduction to keep pH high, [H+] low
c.Why doesn’t BH4 reduce galactose generated by hydrolysis in anthrone reaction?
3.Anthrone Reaction
a.Get variation of color intensity depending on sugar
i.Use sugar as standard that you want to assay (i.e. glucose for glucose, lactose for lactose).
ii.In this case use lactose treated with BH4 exactly as samples
b.As always, don't extrapolate beyond highest standard or dilute sample after reaction. Reagent may have been used up. If you did the assays correctly, all samples will be lower than highest standard.
c.Cautions: Anthrone reagent highly acidic!
i.Wear goggles
ii.Use Spec 70s because we don’t want acid near computers.
iii.If you spill, clean up promptly using NaHCO3. Tell instructor
iv.Dispose of reagent in appropriate bottle in hood.
4.Controls
a.For glucose oxidase use lactose--why?
b.For lactose use glucose--why?
D. Calculations
1.
2.For glucose or lactose
a.
b.
3.Plot moles sugar/ml medium vs. time. First time point for each sugar should be 1.5 mM (= 1.5 µmole/ml)
II.Problems--these are old exam questions
1. Design an experiment that will allow you to directly correlate the apparent A600 of a bacterial culture measured on a particular spectrophotometer with the actual number of cells in the culture.
Solution: Since each spectrophotometer will measure the OD (light scattering rather than absorbance) somewhat differently, you must calibrate the instrument you are using. There are a couple of ways you can do this. 1. Grow a culture of the bacterium you are interested in. Measure the OD of the growing culture at several time points and at the same time make dilutions of the culture and plate on agar plates. Incubate overnight and count colonies. Calculate (using the dilution factor) to determine the concentration of cells that gave the OD you observed. Plot OD (y axis) vs. cell count (x axis). This standard plot can then be used to determine the cell concentration in a growing culture by measuring the OD. 2. Grow cells in the same way. Take the OD and at the same time, dilute the cells and count them in a counting cell under a microscope. Note that method 1 will give only live cells while method 2 will give all cells.
2.Outline a method of assaying the amount of glucose and lactose found in a culture medium that can be performed using only enzymes and enzyme assays that we have used in class (acid phosphatase, glucose oxidase, peroxidase, GOT, MDH, -galactosidase, polymerase, ligase, various endonucleases). Be sure your answer explains how to distinguish between glucose and lactose and how to quantitate each sugar. You cannot use the anthrone test or borohydride reduction to answer this question.
Solution:The problem here is to distinguish between the two sugars. You can determine the [glucose] easily using the glucose oxidase/peroxidase enzymes of the Sigma Glucose Assay Kit (and the o-dianisidine). Lactose is a -galactoside, so it will be hydrolyzed by -galactosidase, but the hydrolysis doesn't produce a color. You can hydrolyze the lactose with -galactosidase and then run the glucose oxidase/peroxidase reaction to get a colored product (actually you can do them all at the same time in a coupled reaction). The reaction with just glucose oxidase/peroxidase will measure [glucose] only. The reaction with -galactosidase in addition to glucose oxidase/peroxidase will give the total of [glucose] + [lactose]. By subtracting the [glucose] from {[glucose] + [lactose]} you will have the [lactose].
3.You are studying the effects of a new drug treatment for diabetes. To monitor the drug' s effects, you draw blood from test subjects and measure glucose (MW 181 g/mole) concentration in the blood. For each sample 5.00 ml of blood serum is diluted with 10.00 ml of PBS and then assayed as follows:
1.00 mg/ml Glucose(ml) / PBS
(ml) / Assay
Rgt (ml) / A525 / [glucose]
(units?) / Diluted Serum
(ml) / PBS (ml) / Assay
Rgt
(ml) / A525 / [glucose]
(units?)
0.000 / 1.000 / 5.00 / 0.000 / 0.200 / 0.800 / 5.00 / 0.215
0.200 / 0.800 / 5.00 / 0.190 / 0.500 / 0.500 / 5.00 / 0.575
0.400 / 0.600 / 5.00 / 0.365 / 1.000 / 0.000 / 5.00 / 0.995
0.700 / 0.300 / 5.00 / 0.655
1.000 / 0.000 / 5.00 / 0.895
a.Calculate the glucose concentration or amount in each standard tube. Show sample.
b.Graph the standard curve
c.Determine the concentration of glucose in the blood in mg/ml and in mM
d.Calculate the molar extinction coefficient of glucose in this assay.
Solution:
a.Sample [glucose] calculation: (0.200 ml)(1.00 mg/ml) = (6.00 ml)(x mg/ml)
x = 0.0333 mg/ml
Sample glucose amount calculation: (0.200 ml) (1.00 mg/ml) = 0.200 mg glucose
b.Standard curve
c.Using amount:
Reading from the standard curve
sample a: 0.222 mg glucose in 0.200 ml; 0.222 mg/0.200 ml = 1.11 mg/ml
sample b: 0.624 mg glucose in 0.500 ml; 0.624/0.500 = 1.248
sample c is off the standard curve
Averaging a and b gives 1.18 mg/ml (3 sig. figs.) glucose in the DILUTED sample
Calculate the undiluted concentration: (1.18 mg/ml)(15 ml total volume/5 ml serum) =
3.54 mg/ml;(3.54 mg/ml)(1 mmole/181 mg) = 0.0196 mmol/ml
= 19.6 mmol/liter =19.6 mM
Using concentration:
Reading from the standard curve
sample a: 0.0370 mg/ml; (0.0370 mg/ml)(6.00 ml) = (0.200 ml)(x)
x = 1.11 mg/ml
sample b: 0.104 mg/ml; (0.104 mg/ml)(6.00 ml) = (0.500 ml)(x)
x = 1.248 mg/ml
sample c is off the standard curve
Averaging a and b gives 1.18 mg/ml (3 sig. figs.) glucose in the DILUTED sample
Calculate the undiluted concentration: (1.18 mg/ml)(15 ml total volume/5 ml serum) =
3.54 mg/ml;(3.54 mg/ml)(1 mmole/181 mg) = 0.0196 mmol/ml
= 19.6 mmol/liter =19.6 mM
d.Molar extinction coefficient: You must use final concentration to do this calculation!
Use one point on graph--I chose (0.365, 0.0667 mg/ml)
A = E•C•l
0. 365 = E •0.0667 gm/ml •(1.00 mmole/181 mg)•1.00 cm
E = 990. ml/mmol•cm ( = 990. liter/mole•cm)