EXPERIMENT 4: Enzymes

IB. The protein nature of enzymes

IA. Preparation of catalase
Grated Potato (w/ skin) 5mL Extract 5mL Extract
+ 5mL 6% H2O2 + 5mL 3M NaOH

Extract

+ lighted matchstick + 5 dp 0.1 M CuSO4

Juice Pulp +H2O
Note what happened & for the gas Note color change

Strain Extract

Extract Discard pulp

II. Factors that affect enzyme activity

Test for complete Hydrolysis: in spot plate
Effect of pH

#1 #2 #3 2dp soln (fr 3 test tubes)

10 mL starch soln
+ 1 mL 0.1M NaCl
+ 1mL 0.05 N HCl
+2 mL saliva
10 mL starch soln 10 mL starch soln +2 dp iodine soln (every 3 mins)
+ 1 mL 0.1M NaCl + 1 mL 0.1M NaCl
+ 1mL H2O + 1mL 0.05 N NaOH
+2 mL saliva +2 mL saliva
+ lighted matchstick

H2O bath (37C) If blue, continue iodine dps every 3 mins.

Note time of complete hydrolyzation

Test for complete Hydrolysis: in spot plate

Effect of Temperature

#1 #2 #3
10 mL starch soln 10 mL starch soln 10 mL starch soln
+ 1 mL 0.1M NaCl + 1 mL 0.1M NaCl + 1 mL 0.1M NaCl
+2 mL saliva +2 mL saliva +2 mL saliva

H2O (room temp) H2O bath (37C) H2O bath

Test for complete Hydrolysis: in spot plate

 EXPERIMENT 1: Carbohydrates: Extraction and Reactions
I. Preparation of Sample (Starch) & General Test

Sucrose, Glucose, Fructose, Lactose

Grated SAMPLE

+100 mL H2O
MOLISCH TEST
Filtration
2mL Sample
Residue Filtrate (Liquid)
+ 10 dp Molisch
Wash, Decantation + 15-20 dp H2SO4
Strain & collect Starch
+ 5 mL H2O (dilute)
Note color change at
junction & Record

IODINE TEST
Test for Carbohydrates
(+) Purple @ junction
2mL Sample
+ 1 dp iodine soln

Note color change & Test for polysaccharides

Record (+) Blue-black color
(-) Yellow orange

II. Specific Reactions

3mL sugar SAMPLE Sucrose, Glucose, Fructose, Lactose

OSAZONE SELIWANOFFs TEST FEHLINGs TEST BARFOEDs TEST BENEDICTs TEST

+1msp Phenylhydrazine 2mL Sample 1mL Fehlings A&B 2mL Barfoeds 2mL Benedicts
+ 10dp con. Acetic acid + 3mL Seliwanoff + 3mL H2O + 10 dp Sample + 15 dp Sample
(CH3COOH)
15 mins 1 min 5 mins 2 mins
+ 1/4 msp Sodium acetate
(C2H3NaO2) Note color change & +8 dp Sample Note color change w/in Note color change &
2 mins
Record color & time 15 mins. & Record Record
10 mins
5 mins
Note color change &
Record
Note Crystals in
Microscope

Test for reducing suaars Test for ketose Test for aldehydes Test for mono Test for reducing sugars
(+) Cherry red complex (+) Reddish brown ppt (+) brick red ppt (+) Red orange soln

LEGEND:
Heat dp Drop Mix Ice bath
Bench Cool Incline Do not Stir
 EXPERIMENT 1: Carbohydrates: UNKNOWN
UNKNOWN SAMPLE

se, Fructose, Lactose 2mL Sample

MOLISCH TEST + 10 dp Molisch
+ 15-20 dp H2SO4

(-) Non- Carbohydrate (+) Purple @ junctionCarbohydrate

IODINE TEST 2mL Sample

+ 1 dp iodine soln

(-) Yellow orange mono/di

2mL Sample SELIWANOFFs TEST (+) Blue-black color

STARCH polysaccharide
+ 3mL Seliwanoff
15 mins

(-) Ketose (+) Cherry red complex Ketose

FEHLINGs TEST BENEDICTs TEST

1mL Fehlings A&B 2mL Benedicts

+ 3mL H2O + 15 dp Sample
1 min 2 mins

+8 dp Sample
2 mins
(-) SUCROSE
(+) Red-orange soln
Non-reducing Aldo-ketose di
FRUCTOSE
(-) LACTOSE (+) Reddish-brown ppt Reducing Aldo-ketose di
Aldose di GLUCOSE
Aldose mono
EXPERIMENT 3: Properties of Lipids
I. Solubility
1dp coconut oil

+1 mL H2O +1 mL chloroform +1 mL CCl4 +1 mL C6H14 (Hexane) +1 C2H6O (EthylOH) +1 mL H2O

Note for solubility:

Degree: very soluble, slightly soluble, insoluble

II. Emulsification
1mL coconut oil
+ 3mL H2O

+1 mL 0.5%Na2CO3

Note changes

III. Acrolein Test

presence of glycerol or fat

10 dp Castor oil 10 dp glycerol 10 dp oleic acid

+ 1microspatula KHSO4

Note changes

IV. Test for Unsaturation

1mL Chloroform

+ 1dp cottonseed oil +1 dp linseed oil +1dp oleic acid

+ Hubls soln
(dp by dp + shake)

Note color change &

Record color & # of dps
EXPERIMENT 2: Color Reactions of Proteins
BIURET TEST

1 mL 10% NAOH
+3mL sample Note color change

+ 1 dp 0.010M CuSO4 (if needed, +1 dp 0.010M CuSO4

NINHYDRIN TEST

5 dp 0.15 Ninhydrin Note color

+ 2 mL sample 10 mins change

XANTHOPROTEIC TEST

1mL conc. HNO3 + sat. NaOH Note color

+ 3mL sample Cool (until alkaline/basic) change

MILLONS TEST

5 dp Millons reagent Note color

+ 3mL sample 5 mins change
cool

HOPKINS-COLE TEST

2mL Hopkins-cole r. Note color change

+ 3mL sample after 1-2 mins
+ H2SO4

SAKAGUCHI TEST

1ml 10% NaOH Note color

+2-4 mL NaOCl
+1 Ml 0.02% napthol 3 mins change
+ 3mL sample

LEAD ACETATE TEST

5mL 5% NaOH Note color

+ Pb(Ac)2 5-10 mins change
& stir
+ 3 mL sample