SUBJECT: Benedict's solution/amylase
DATE: 2/95
We've been using Benedict's to show the presence of sugar after
starch digestion by amylase.
For the first time, we're getting results that don't make sense -
i.e. digestion after boiling & no change with temperature.
I checked everything & repeated everything with the same results.
I tried both bacterial & pancreatic amylase with the same results.
In desperation, I finally combined only amylase and Benedict's and
got a positive result for sugar!
Any idea what's happening? Thanks in advance.
Lynn
********************************************************
Lynn Ruxton (Lynn.Ruxton@lakeheadu.ca)
Biology, Lakehead University,
Thunder Bay, Ontario, Canada P7B 5E1
807-343-8593 FAX:807-346-7796
At least some amylases are glycoproteins and could
conceivably give a positive benedict's test, but you shouldn't
be using that much enzyme. Check your source- does the
enzyme have a high specific activity (i.e., few
contaminants)? Some preparations have stabilizers added
that might cause the reaction. Dialysing the enzyme solution
should remove them.
As for your experiment itself, the enzyme might not be the
only problem. Does your substrate test negative without
enzyme? I've had problems on occassion when microbial
growth in my substrate solution digests part of the starch
before we even add the enzyme!
John Dickerman
Northern Illinois University
DeKalb, IL 60115
T80JWD1@WPO.CSO.NIU.EDU
Re replies to Benedict's query:
I tried boiling just Benedict's & starch & got a negative result
so I
assume that neither the starch solution nor the Benedict's are
contaminated with sugar.
The bacterial amylase is from Carolina & there was no information on
the bottle other than its name. It gave a very strong positive
result to the Benedict's. The pancreatic amylase I made fresh from
powdered pancreatin & its response wasn't quite as strong a positive
as the bacterial amylase gave.
Can Benedict's solution itself degrade with time so the enzyme can
attack it & cause a precipitation?
Thanks again.
Lynn
********************************************************
Lynn Ruxton (Lynn.Ruxton@lakeheadu.ca)
Biology, Lakehead University,
Thunder Bay, Ontario, Canada P7B 5E1
807-343-8593 FAX:807-346-7796
**************************************************
I had so many of these type of problems, that I started using "time
to %100
hydrolysis" with iodine instead of showing sugar. It always works.
--------------------------------------------------------------------------------
Thomas Pitzer Office: OE 296 Phone: (305) 348-1224
Instructor/Coordinator Undergraduate teaching Fax: (305) 348-1986
Internet: pitzert@servms.fiu.edu
Florida International University Dept. Biological Science--OE 246
University Park Miami, FL 33199
--------------------------------------------------------------------------------
Benedict's reagent is pretty much a basic solution of copper
sulfate- the aldehyde in the sugar reduces the Cu++ ion to
copper atoms. The most obvious decay here would be
for the solution to pick up CO2 over time and neutralize
the basicity, but that should lead to no positive tests. Any
way, if you want to try it here's a recipe for Benedict's
reagent from Carolyn Eberhard's "Experiments in Biology":
Dissolve 173g of sodium (or potassium) citrate and 200g of
sodium carbonate (NOT bicarbonate!) in 800 ml of distilled
water. Dissolve 17.3 g of CuSO4 in 100 ml of distilled water.
Mix slowly and bring to 1 liter. (Don't mix everything
together at once or it will never dissolve.)
Good luck!
John Dickerman
Northern Illinois University
DeKalb, IL 60115
T80JWD1@WPO.CSO.NIU.EDU
I agree with Tom Pitzer that using time to 100% hydrolysis of starch
always works. I do have the students test for sugar at this point -
but they use "Clinitest" tablets (available commercially to test
for
sugar in urine in diabetics) instead of the Benedict's test. The
control tube (students boil the amylase before adding starch) is
almost always negat ive for sugar when they use this test for sugar.
Lucy Dyer
Biology Department, U.N.B., Fredericton, N.B.