• Table 1: Calibration curve for reducing sugar

• Table 2/Experiment A: The effect of enzyme concentration on initial velocity

• Table 3/Experiment B: The effect of the length (time) of incubation on product formation.

• Table 4/Experiment C: The effect of substrate concentration on the rate of the normal catalysed reaction

• Table 5/Experiment D: The effect of substrate concentration on the rate of the urea inhibited reaction
 Calibration curve for reducing sugars

Tube # Vol. of glucose standard (mL) Abs. @ 510 nm μmol of reducing sugar
1 0.00    
2 0.05    
3 0.05    
4 0.10    
5 0.15    
6 0.20    
7 0.20    
8 0.25    
9 0.30    
10 Fructose (4mM, 0.2mL)    
11 Sucrose (4mM, 0.2mL)    
Table #4Enzyme Activity of Fraction 4

Additions SUCROS GLUCOSE GLUCOSE FRACTION 4 ZERO

E BLANK STANDARD TIME
BLANK CONTROL
Tube # 1 2 3 4 5 6 7 8 9 10
Acetate Buffer 0.2 --- --- 0.2 0.2 0.2 0.2 0.2 0.2 0.2
Deionized Water 0.6 1.0 0.8 0.58 0.55 0.5 0.4 0.2 --- ---
Volume of --- --- --- 0.02 0.05 0.1 0.2 0.4 0.6 0.6
Fraction 4
Volume of --- --- 0.2 --- --- --- --- --- --- ---
glucose 4mM
  START REACTION BY ADDING SUBSTRATE
Vol. of 0.5M 0.2 --- --- 0.2 0.2 0.2 0.2 0.2 0.2 0.2
Sucrose
  Incubate all tubes for 10 minutes at room temperature. Stop the assay by addition of Nelson’s reagent. VORTEX!
Nelson’s reagent 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
  Place the tubes in a boiling water bath and continue with the Nelson’s procedure for reducing sugars. VORTEX!
Absorbance at 0.00 0.00                
510nm
μmol reducing                    
sugar in 10 min.
μmol/min/mL                    
 
Avg. μmol/min/mL          
 
Units/mL stock          
fraction
1.  
umol of reducing sugar/min : Session 4 - The sample calculations for Experiments A and Exp B – only
umol of reducing sugar required.
Exp. A (+ B) – absorbencies must be corrected using the appropriate zero-time
control
Assume the equation for the Nelson’s calibration curve was y = 0.8153x
Using a corrected absorbance of 0.025, umol of reducing sugar for 0.02 mL aliquot (Exp A) = 0.025/0.8153
= 0.031 umol.
 
• umol/min = 0.031/10 min = 0.0031umol/min
• umol/min/ml = answer
•  NB at some point must divide by 2 because we are assessing activity in moles of sucrose broken
down, not umol of reducing sugar formed.
• Then find average for all 5 aliquots
• Then 1 in 1000 dilution of fraction 4 was done. Therefore you find the undiluted stock
 A. The Effect of Enzyme Concentration on Initial
velocity
 
Tube# Volume of Fraction 4 Abs. at 510nm Corrected Abs. at mg of protein μmol reducing (v)
510nm sugar μmol/min

1 SUCROSE BLANK          
2 GLUCOSE BLANK          
GLU. STANDARD (0.2ml)          
3

4 0.02          
5 0.05          
6 0.10          
7 0.20          
8 0.40          
9 0.60          
0.60          
10 (Zero-time control)
2. For Exp. A - enzyme content to plot for graph 2.

Even though this is not completely accurate we will take

the total protein in the aliquot to represent the amt. of
enzyme in the sample. This would be from session 3
 
Assume equation of the line from session 3  y = 0.08x
For 0.02 mL fraction corrected abs = 0.034
mg of protein = 0.034/0.08 = 0.425 mg and so forth.
3 Units of enzyme activity per mL of original fraction 4.
 
Assume an arbitrary value of 82 U/ml s obtained from table 4
R.T.F. 0.1mL yields 1 μmol/min

1mL will be equal to 10 μmol/min

Conc. of stock F4= 82 U/ml

Using C1V1 = C2V2

(82 μmol/min/mL) (x) = (10μmol/min/mL) (5mL)

x = (10 x 5)/82 mL
= 0.61 mL
 Table #5 The Effect of Incubation on Product Formation
Additions (mL)     Control Glucose Glucose
FRACTION 4 without blank standard
enzyme
Tube # 1 2 3 4 5 6 7 8
Zero Table #5The Effect of 9Incubation on
11 12
Product Formation 10

timea aNelson’s reagent is added before the enzyme to this tube.

Acetate Buffer 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 --- ---
bEnzyme should be diluted so that 0.1mL will yield 1μmol/min under normal assay conditions
0.5M Sucrose 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 --- ---
(diluted 1:25)
 
Deionized Water 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.7 1.0 0.8
  START ASSAYS BY THE ADDITION OF ENZYME --- ---
Purified enzymeb 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 --- --- ---
Volume of glucose --- --- --- --- --- --- --- --- --- --- --- 0.2
(4mM)
Incubation Time 0 1 2 4 8 10 12 15 20 20 10 10
(min.)
  Incubate tubes for the designated time intervals. Stop the reaction by the addition of 1 mL of Nelson’s reagent
Nelson’s reagent 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
  Cover the tubes with foil, place in a boiling water bath for 20 min, and assay for reducing sugar as described by
the Nelson’s procedure.
Absorbance at                        
510nm
mmol reducing                        
sugar formed
 B. The Effect of incubation Time on Product Formation

Tube# 1 2 3 4 5 6 7 8 9 10 11 12
                         
Time (min) 0 1 2 4 8 10 12 15 20 20 10 10

Abs. @ 510nm 0.00                   0.00  

                       
μmol reducing
sugar
How does incubation time affect the rate of reaction?
Incubation time. The longer an enzyme is incubated with its substrate,
the greater the amount of product that will be formed. ... As a result,
the rate of formation of product slows down as the incubation proceeds,
and if the incubation time is too long, then the measured activity of the
enzyme is falsely low.
Between A and B, the curve represents a zero
order reaction; that is, one in which the rate is
constant with time. As substrate is used up, the
enzyme's active sites are no longer saturated,
substrate concentration becomes rate limiting, and
the reaction becomes first order between B and C.
To measure enzyme activity ideally, the
measurements must be made in that portion of the
curve where the reaction is zero order. A reaction
is most likely to be zero order initially since
substrate concentration is then highest. To be
certain that a reaction is zero order, multiple
measurements of product (or substrate)
concentration must be made.
 Session 5.
NB for this session we have to dilute the column fraction
1 in 1000. We were supposed to do this and then based
on graph 2, from Enzyme concentration part A session 4.
The amount of enzyme added should be non-limiting i.e.
about 2/3 up the linear portion of the enzyme
concentration curve .

Let us assume that correlated to 0.1mL for simplicity.

You can indicate on your graph the linear portion
 TABLE #7 Effect of Substrate Concentration on Enzyme Activity

Glucose
BLANK Fraction 4 Sucrose correctiona Glucose blank
standard
Tube# 1 2 3 4 5 6 7 8 9 10 11 12

0.2M acetate
0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 --- ---
buffer, pH 4.5

0.5M Sucrose
--- 0.02 0.04 0.06 0.08 0.1 0.2 0.4 0.2 0.4 --- ---
(mL)
Deionized
0.7 0.68 0.66 0.64 0.62 0.6 0.5 0.3 0.5 0.3 1.0 0.8
water
Enzymeb 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 --- ---
Glucose
--- --- --- --- --- --- --- --- --- --- --- 0.2
(4mM)

Nelson’s
1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
reagent

A510nm 0.00 0.00

μmol reducing
sugar/min
Exp. C + D – sucrose correction (i.e. non-
enzymatic hydrolysis) is somewhat involved

Used 0.2 and 0.4 mL (tubes 9 and 10) sucrose

aliquots, obtained 2 absorbance values.
Standardize absorbencies to e.g. 1 mL and then
find average. For each sucrose aliquot used
in the assay, use the average absorbance per ml
to find what the absorbance would have been if
you had included a zero-time control for each
tube. Take this absorbance away from the
actual tube absorbance to get the corrected
absorbance 
5. Sucrose concentration per tube – Experiment C + D
 
Conc of sucrose = 0.5M, i.e. 0.5 mol/1000mL
 no of mol in e.g. 0.02 mL = (0.02 x 0.5)/1000 = 1 x 10-5 mol or
0.01 mmol.
 
Total assay volume = 1 mL
\ conc. of sucrose in tube = 0.01 mmol/mL or 10 mmol/L i.e.
10 mM.

From tables you are required to find for both uninhibited and
inhibited rxns :

micromoles of reducing sugar /min = v

7. Km and Vmax = Read off values from
Michaelis-Menten curve (must indicate
points on graph). Lineweaver-Burk plot –
find the negative inverse for -1/Km and the
inverse for 1/Vmax  Must show these
points as well on graphs (Both inhibited
and uninhibited reactions).
 C & D: The Effect of Substrate Concentration and Inhibitor on Enzyme Activity

Volume of sucrose (mL) Concentration of sucrose (M) Abs. at 510nm Abs. at 510nm with Urea
without Urea
0.00      
0.02      
0.04      
0.06      
0.08      
0.1      
0.2      
0.4      
0.2      
0.4      
Glucose      
blank
Glucose standard 0.2mL of 4mM standard    
6. Calcfor 1/V and 1/[S]
Velocity would of course be the inverse of the activity shown in calculation 1 and 1/[S] is
shown in the table immediately above.

NB. The script said to represent velocity as umol of reducing sugar/min, but since Km represents
the affinity of the enzyme for its substrate, shouldn’t velocity be divided by 2?
Effectively everything is still proportional however if one wanted to be exacting,
I think this would have been better.
 5. Summary

Vol of 0.5 M 0.02 0.04 0.06 0.08 0.1 0.2 0.4

sucrose (mL)

[S] mmol/L 10 20 30 40 50 100 200

1/[S] mmol-1L 0.1 0.05 0.033 0.025 0.02 0.01 0.005

• Graph 1: Calibration curve for reducing sugars

• Graph 2: The effect of enzyme concentration on the initial

velocity of reaction. Plot of average umol of reducing sugar
per minute vs. amt. of enzyme –( taking this as the amt. of
protein in the aliquot – based on Bradford assay from session
3).

• Graph 3: The effect of time incubation on product formation

Plot of umol reducing sugar formed vs. time (mins)

• Graph 4: The effect of [S] on reaction rate (average umol of

reducing sugar per minute) for the inhibited and non-
inhibited reactions (Michaelis-Menten curves –must indicate
Vmax and Km for both reactions  

• Graph 5: Lineweaver-Burk plot for the above (both

reactions). Must indicate –1/Km and 1/Vmax for the two
lines.