Dear Ilias,

The following publications depict the substrates that can be used in the mentioned enzymatic reaction:

1-Actually the substrate is ortho-nitrophenyl -beta-glucopyranoside"

The enzyme !-galactosidase catalyses the following reaction:

LACTOSE + beta-galactosidase  = GLUCOSE + GALACTOSE

The chemical ONPG (o-nitrophenyl !-D-galactopyranoside) is also degraded by the enzyme:

ONPG + beta-galactosidase =  ONP + GALACTOSE

The ONP produced is yellow, allowing the rate of this reaction to be followed colorimetrically.

Galactose acts as a competitive inhibitor, competing with ONPG for the active site of the enzyme. At a sufficiently high concentration, it will inhibit the reaction by preventing ONPG making contact with the active site. The enzyme, however,

is still capable of activity. Thus, when the ONPG concentration is increased it will eventually overcome the inhibition.

Iodine solution on the other hand is a non-competitive inhibitor. When it combines with the enzyme the shape of the active site is altered sufficiently to prevent the substrate combining with it. Increasing substrate concentration will therefore not overcome the inhibition.

References

1-Adds, Larkcom and Miller (eds.), (1996) Cell Biology and Genetics, Nelson Advanced Modular Science, ISBN 0–17–448266–3

2-Hames, B.D., Hooper, N.M. and Houghton, J.D. (1997) Instant Notes in Biochemistry, Bios Scientific.

3-Russo, S. E. and Moothart, L. (1986) Kinetic study of the enzyme lactase. Journal of Chemical Education, 63(3), 242–243.

2-Product name Beta Galactosidase Detection Kit (Fluorometric)

Sample type Adherent cells, Suspension cells

Assay type Cell-based (quantitative)

Sensitivity >= 0.3 mU/well

Assay time 0h 30m

Species reactivity Reactswith: Human

Predicted to work with: all Mammals

Product overview Beta Galactosidase Detection Kit (Fluorometric) (ab176721) uses the fluorogenic fluorescein digalactoside (FDG) galactosidase substrate that can sensitively distinguish LacZ+ from LacZ- cells. The non-fluorescent substate

generates the strongly fluorescent fluorescein upon reaction with galactosidase. It can be used either for detecting galactosidase conjugates in ELISA type assay systems or for monitoring LacZ gene expression in cells. FDG used in the kit is not fluorescent. The galactosidase induced cleavage of FDG gives fluorescein that

has the spectra of Ex/Em = 490/515 nm, which can be detected with most fluorescence instruments equipped with a FITC filter set. The kit comes with all the essential components with an optimized assay protocol. It can be used with a fluorescence microplate reader or a fluorescence microscope. It might also be used for screening galactosidase inhibitors or inducers.

Tested applications Functional Studies

Relevance Beta galactosidase is a hydrolase enzyme that cleaves beta-linked terminal galactosyl residues from gangliosides, glycoproteins, and glycosaminoglycans. Beta galactosidase is an essential enzyme in the human

body. Deficiencies in the protein can result in galactosialidosis or Morquio B syndrome. Senescent cells display senescence-associated expression of beta galactosidase activity.

Cellular localization Isoform 1: Lysosome. Isoform 2: Cytoplasm, perinuclear region. Note=Localized to the perinuclear area of the cytoplasm but not to lysosome

For more details, see the attached file

3-Chemiluminescent b-Galactosidase Protocols

See attached file

Hoping this will be helpful,

Rafik

Do i can use Lactose to generate : Glucose and Galactose

and use DNS for quantification, since i do not possess ONPG ?

Dear Ilias,

To some extent yes since there are some limitation; please read the following:

The hydrolytic conversion of lactose to glucose and galactose represents one way ofadding value to whey and whey-derived products. For the enzymatic lactose hydrolysis various mesophilic �-glycosidases have been described, some of which have already made it to the market. The application of known glycosidases is however partly hampered because of the moderate thermal stability and narrow pH profile of enzyme activity as well as due to the significant inhibition by galactose. The use of hydrolases from thermophilic microorganisms could help to overcome at least some of these problems.

http://www.rpi.edu/dept/chem-eng/Biotech-Environ/IMMOB/lactose.html

Hoping this will be helpful,

Rafik

Hi there,

ONPG has the advantage to allow direct and continuous assay by following OD increase at 410-415nm.

Yes for sure ONPG is the best choice 

However the reagents in my possession allow me to use a protocol that i have found in previous paper.

I am not sure if i can use it for my assay to evaluate inhibitory properties:

b -galactosidase : Lactulose ==== fructose + galactose 

MTT+ FDH: d -fructose  ==== 5-keto-d -fructose + MTT Formazan

You can find the paper attched

I don't see any problem using this protocol as lactulose is a substrate of beta-galactosidase. The only thing to do is to add the inhibitor during the assay and make sure you are actually measuring initial velocities.

Dear Ilias, can you check these documents please, maybe they could help you

Good Luck

Hi Ilias,

 If you are to use ONPG or PNPG, the yellow coloration will only form in alkaline solution. Thus, you can use 0.10M NaOH. This will inhibit the enzyme activity (thus stopping the reaction when desired) as well as develop a yellow coloration that can be read between 400-410nm.