I'm checking the inhibition of amylase enzyme through starch hydrolysis. I will explain the protocol here:
Reagents:
PBS buffer- 20 mM, 6.9 pH, with 0.1 mM Cacl2 and 6.7 mM Nacl
40 U amylase
50 mg corn starch
0.5 mol/L sodium carbonate
Procedure:
1. 50 mg corn starch + inhabitor (3 different ratios- 1:0 (control), 1:2, 1:4, 1:6) + 9 ml PBS buffer incubated for 15 mins at 37 C with continuous mixing
2. After 15 mins, 1 ml amylase (1 ml containing 40 U) was added.
3. 300 uL of the mixture was withdrawn in predetermined time intervals (30 mins) - from 0th, 240th min to a centrifuge containing 1.2 ml sodium carbonate (stopping solution)
4. The sample mixtures were centrifuged for 5 mins at 11,800 g and 100 uL of supernatant was transferred to another centrifuge tube followed by the addition of 300 ul DNS reagent.
5. The tubes were boiled at 90 C for 5 mins.
A maltose std was also plotted. (45 mg in 50 ml DW) Concentrations taken- 0, 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, 1 ml volume was made up to 2 ml and 1 ml DNS reagent was added.
My query:
DNS reagent reacted well with maltose std solutions and gave darker colours at higher concentrations of maltose.
However, there was no colour change for my samples.
Should I have gelatinized the starch samples before initiating the reaction?
Or the substrate concentration is too less for the enzyme?
Or more DNS reagent needs to be added to the mixture followed by 10 min of incubation in boiling water bath?
I tried adding 1 ml of DNS to 100 uL of the mixture and incubated at 100 C for 5 mins, still no colour change.
It's possible that there is no active amylase present and also that gelatinized starch would not help much because it is reducing sugars which react to develop the requisite red coloration with alkaline DNS reagent.
Arun Padhye thankyou for your response sir. My question is if gelatinization will help in better hydrolysis of the starch? and in my reference paper, they have used PAHBAH (4-hydroxybenzoic acid hydrazide) as the detection reagent. is it more sensitive than DNSA?
Dear Nethra P V,
Obviously, gelatinization would facilitate starch hydrolysis. Whether it is acid hydrolysis or enzyme facilitated starch hydrolysis the gelatinization is a prerequisite for hydrolytic degradation of starch.
Native starch granules resist hydrolysis due to densely packed amylose and amylopectin molecules through hydrogen bonding within the granule case.
I am not very sure as to which reagent is more sensitive relatively amongst DNSA and PAHBAH. If my wild guess is right then it is the latter which is probably more sensitive.
The explanation by Arun Padhye is correct. Enzymatic hydrolysis of ungelatinized native starch granules is very slow, due to the semi-crystalline nature of intact starch granules. Gelatinization is best achieved either by (stirred) heating in excess water (ca 90 C) or by stirring in 1 M NaOH followed by pH adjustment with aqueous HCl. To prevent lumping or sticking I recommend to prepare a stock solution of gelatinized starch by suspending the granular starch in 1 volume of water, add 1 volume 2 M NaOH (all with stirring) and neutralize with aqueous HCl to desired pH. For doing the assays, aliquots are taken and buffer, inhibitor, and amylase are added.
Arun Padhye Peter A. M. Steeneken yes sir, thankyou for your suggestions. I tried them out and gelatinized starch did give me colour with DNSA.
@Netra P V, Take care of one additional blank when you read absorbance of red color samples. This is needed because when you use gelatinized starch during acid/enzyme hydrolysis some reducing sugars including glucose and maltose etc. are formed and you need to substract this effect to increase the assay accuracy.
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