Qualitative Color Reactions

Color Reaction

Intact
Protein

Biuret

Protein Hydrolysate
acidic

basic

enzymatic

Ninhydrin

Xanthoproteic

(+) Y

+/(++) F/Y

+
(FYW)

+ (C,Pe,B,Pa)

Millons

(+++)

+/-

+ (C,Pe,B,Pa)

Hopkins-Cole

+ (C,Pe,Pa)

Sakaguchi

(+++)

+ (T,Pa)

Nitroprusside

+/-

Fohls

+
-

+/-

Test for amide

Pauly

+ (C,Pe,B,Pa)

Beer lamberts law

relate the absorbance of a solution to the
concentration of a particular solute in that
solution

Enzymes

Invertase
Yeast derived enzyme
Official name: -fructofuranosidase (EC
3.2.1.26)
Classified as hydrolase catalyzing hydrolysis
of the terminal nonreducing fructofuranoside residues
Splits sucrose to glucose and fructose

Mechanism of Reaction

-D-glucose + -D-fructose

Dinitrosalicylic Acid Assay

Dinitrosalicyclic acid (DNS) Assay
this method is used to monitor
enzyme activity.

Principle Involved:
DNS (3,5-dinitrosalicylic acid, IUPAC
name 2-hydroxy-3,5-dinitrobenzoic
acid) reacts with reducing sugars
(eg. glucose and fructose) to form
3-amino-5-nitrosalicylic acid (ANS).
DNS does not react with sucrose
(non-reducing sugar).

Dinitrosalicylic Acid Assay

In the DNS assay, rate of reaction (enzyme activity) is
monitored (colorimetrically) by measuring the
amount of reaction products (reducing sugars equimolar mixture of glucose and fructose) that
react with DNS reagent.

Dinitrosalicylic Acid Assay

Reagents for DNS assay
Conc HCl for complete hydrolysis of glycosidic
bonds
0.5N KOH to neutralize excess acid
1% DNS reagent
Dinitrosalicylic acid oxidizing agent
Na2SO3 stabilizes the red color
NaOH increases the reactivity of sugars; changes the
pH of the reaction vessel (along with ANS production)
halting the invertase reaction.

Factors Affecting Enzyme Activity

pH
pH dependence of enzyme activity is a
consequence of acid-base behavior or
changing degree of ionization of groups in the
enzyme, in the substrate, or in both.
Invertase exhibits high activity over a broad
pH range 3.5-5.5 with optimum pH near 4.5.

Factors Affecting Enzyme Activity

Temperature
Rate of reaction increases with temp; within
the temp range in which the enzyme is stable
and retains its full activity. When temp range
is beyond 10-50o at optimum, enzymes are
denatured followed by a decrease in enzyme
activity.
Optimum temp of invertase = 55oC

Factors Affecting Enzyme Activity

Enzyme concentration (when [S] is constant)

Substrate concentration (when [E] is constant)
Enzyme inhibitors/activators
Cofactor/Coenzyme