Set up a colorimetirc assay for alpha-glucosidase activity so that you can perform a continuous assay. This makes it easy to measure the initial rate of each reaction. It is crucial to use only initial rates. This means you have to measure the slope of the earliest part of the progress curves, so they must be linear at the start.

Perform a matrix of experiments in which you vary both the alpha-glucosidase substrate concentration and the inhibitor concentration. About 6 concentrations of each covering the dynamic range should be sufficient. Measure the initial rate for each reaction. Do it in triplicate, if possible. This can be done quite quickly in 96-well plates if you have an absorbance plate reader.

Fit all the data at once to the Michaelis-Menten equations for competitive, non-competitive, and uncompetitive inhibition mechanisms using a nonlinear least-squares regression computer program. If one equation fits much better than the other (lowest chi-squared), that is the mechanism of inhibition.

If you don't have access to the nonlinear least-squares program, you can use the double-reciprocal plot method, as described in every introductory biochemistry textbook.

The quality of the data is very important when trying to distinguish between mechanisms. Pay special attention to accuracy and precision to minimize the scatter in the data.

Dear Muhammad,

The following publications cover the answer to your question:

Alpha-glucosidase inhibitors are saccharides that act as competitive inhibitors of enzymes needed to digest carbohydrates: specifically alpha-glucosidase enzymes in the brush border of the small intestines. The membrane-bound intestinal alpha-glucosidases hydrolyze oligosaccharides, trisaccharides, and disaccharides to glucose and other monosaccharides in the small intestine.

Acarbose also blocks pancreatic alpha-amylase in addition to inhibiting membrane-bound alpha-glucosidases. Pancreatic alpha-amylase hydrolyzes complex starches tooligosaccharides in the lumen of the small intestine.

Inhibition of these enzyme systems reduces the rate of digestion of carbohydrates. Less glucose is absorbed because the carbohydrates are not broken down into glucose molecules. In diabetic patients, the short-term effect of these drugs therapies is to decrease current blood glucose levels: the long-term effect is a small reduction in hemoglobinA1c level.

1-Clin Invest Med. 1995 Aug;18(4):303-11.

The mechanism of alpha-glucosidase inhibition in the management of diabetes.

Bischoff H1.

Author information

Abstract

The development of the alpha-glucosidase inhibitor acarbose provides a new approach in the management of diabetes. By competitive and reversible inhibition of intestinal alpha-glucosidases, acarbose delays carbohydrate digestion, prolongs the overall carbohydrate digestion time, and thus reduces the rate of glucose absorption. After oral administration of acarbose, the postprandial rise in blood glucose is dose-dependently decreased, and glucose-induced insulin secretion is attenuated. Because of diminished postprandial hyperglycemia and hyper-insulinemia by acarbose, the triglyceride uptake into adipose tissue, hepatic lipogenesis, and triglyceride content are reduced. Therefore, acarbose treatment not only flattens postprandial glycemia, due to the primary and secondary pharmacodynamic effects, but also ameliorates the metabolic state in general. In diabetic animals, acarbose reduced urinary glucose loss, the blood glucose area under the curve, and prevented the decrease in skeletal muscle GLUT4 glucose transporters. As a consequence of the reduced mean blood glucose area under the curve, the amount of protein nonenzymatically glycated was diminished, as was the formation of advanced glycation end-products (AGEs). The prevention of basement membrane glycation and thickening in various tissues indicated that acarbose treatment of diabetic animals produced beneficial effects against the development of nephropathy, neuropathy, and retinopathy. Thus, the alpha-glucosidase inhibitor acarbose may have the potential to delay or possibly prevent the development of diabetic complications.

http://www.ncbi.nlm.nih.gov/pubmed/8549017

2-Eur J Clin Invest. 1994 Aug;24 Suppl 3:3-10.

Pharmacology of alpha-glucosidase inhibition.

Bischoff H1.

Author information

Abstract

The development of alpha-amylase and brush-border alpha-glucosidase inhibitors is reviewed. The mode of action as well as pharmacological and pharmacodynamic properties of selected inhibitors with special regard to the most thoroughly investigated alpha-glucosidase inhibitor acarbose are discussed. Inhibition of intestinal alpha-glucosidases delays the digestion of starch and sucrose, flattens the postprandial blood glucose excursions, and thus mimics the effects of dieting on hyperglycaemia, hyperinsulinaemia and hypertriglyceridaemia. Therefore, the mechanism of alpha-glucosidase inhibition represents the pharmacological optimization of the dietary principle of delayed carbohydrate absorption. In pre-clinical studies using diabetic animals the oral administration of acarbose improved the metabolic state and reduced the blood glucose area under the curve. As a consequence, the process of non-enzymatic glycation of proteins was retarded as indicated by reduced glycated haemoglobin, glomerular basement membranes or advanced glycation end-products (AGEs) in collagen. These improved biochemical parameters correlated with beneficial effects against the development of diabetic nephropathy and neuropathy. Thus, the treatment of diabetic animals with acarbose does not only improve the metabolic state but has also the potential to delay, or possibly prevent, the development of diabetic complications.

http://www.ncbi.nlm.nih.gov/pubmed/8001624

Hoping this will be helpful,

Rafik

Set up a colorimetirc assay for alpha-glucosidase activity so that you can perform a continuous assay. This makes it easy to measure the initial rate of each reaction. It is crucial to use only initial rates. This means you have to measure the slope of the earliest part of the progress curves, so they must be linear at the start.

Perform a matrix of experiments in which you vary both the alpha-glucosidase substrate concentration and the inhibitor concentration. About 6 concentrations of each covering the dynamic range should be sufficient. Measure the initial rate for each reaction. Do it in triplicate, if possible. This can be done quite quickly in 96-well plates if you have an absorbance plate reader.

Fit all the data at once to the Michaelis-Menten equations for competitive, non-competitive, and uncompetitive inhibition mechanisms using a nonlinear least-squares regression computer program. If one equation fits much better than the other (lowest chi-squared), that is the mechanism of inhibition.

If you don't have access to the nonlinear least-squares program, you can use the double-reciprocal plot method, as described in every introductory biochemistry textbook.

The quality of the data is very important when trying to distinguish between mechanisms. Pay special attention to accuracy and precision to minimize the scatter in the data.

Dear Muhammad

Your question is regarding the inhibition mechanism of alpha-glycosidase with specific inhibitor. At this concern, Adam Shapiro wrote an essential draft. In fact you can perform a kinetic study in which initial velocity (v) is determined in the absence/and presence of inhibitor as function of substrate concentration. Then, consider the obtained data, 1/v= f(1/S) to draw curves. Observe the linear curves you obtain and conclude:

(i) Competitive inhibition: generally caused by substrate analogues; the inhibitor fixes in the active site of the enzyme; Km value is modified and becomes greater, enzyme affinity for substrate diminishes apparently.

(ii) Non competitive inhibition: generally caused by inhibitor chemically different of the substrate; the inhibitor site fixation is different of the enzyme active site; the inhibitor fixes on free enzyme and into enzyme-substrate, ES complex. V (comprising Vm) diminishes as there is apparently less enzyme in the assay medium than in the assay control.

(iii) Uncompetitive inhibition (scarce): the inhibitor fixes only into the enzyme-substrate complex (ES) . As a result, the Km and v (comprising Vm) values diminish. This apparently means that in the inhibition process, there is apparently less ES. The inhibition degree increases with substrate concentration increase.

(iv) Very scarce case: the inhibitor behavior does not obey any classical model of inhibition. Then the author search to establish a particular mathematic model in order to calculate Ki (inhibition constant).

It is convenient (recommended) to carry out the studies for at least two different concentrations of the inhibitor.

I hope this comment is helpful.

Dear Muhammad

Your question is regarding the inhibition mechanism of alpha-glycosidase with specific inhibitor. At this concern, Adam Shapiro wrote an essential draft. In fact you can perform a kinetic study in which initial velocity (v) is determined in the absence/and presence of inhibitor as function of substrate concentration. Then, consider the obtained data, 1/v= f(1/S) to draw curves. Observe the linear curves you obtain and conclude (see the attached document):

(i) Competitive inhibition: generally caused by substrate analogues; the inhibitor fixes in the active site of the enzyme; Km value is modified and becomes greater, enzyme affinity for substrate diminishes apparently.

(ii) Non competitive inhibition: generally caused by inhibitor chemically different of the substrate; the inhibitor site fixation is different of the enzyme active site; the inhibitor fixes on free enzyme and into enzyme-substrate, ES complex. V (comprising Vm) diminishes as there is apparently less enzyme in the assay medium than in the assay control.

(iii) Uncompetitive inhibition (scarce): the inhibitor fixes only into the enzyme-substrate complex (ES) . As a result, the Km and v (comprising Vm) values diminish. This means that in the inhibition process, there is apparently less ES. The inhibition degree increases with substrate concentration increase.

(iv) Very scarce case: the inhibitor behavior does not obey any classical model of inhibition. Then the author search to establish a particular mathematic model in order to calculate Ki (inhibition constant).

It is convenient (recommended) to carry out the studies for at least two different concentrations of the inhibitor.

I hope this comment is helpful.

@Adam B Shapiro and Joseph Kreit 

I need your suggestion about selection of sample  and substrate concentrations. As I have already performed alpha-glucosidase inhibition activity for my samples (compounds isolated from plant) in END POINT assay using PNGP as substrate. So I should select my sample concentration around IC50 value and the substrate concentration in higher and lower values than I used in END POINT assay? During kinetic experiment what time interval I should keep for taking repeated absorbance in microplate reader? (In my end point assay incubation time was 40 minutes @ 37 degree C)

Regarding the selection of the time interval for kinetic measurements of initial rates: If you have access to a 96-well absorbance plate reader with kinetic reading capability, then you can just take time points as closely spaced as the reader will allow. For example, a plate reader may require 10 seconds to read all the wells, so your time points can't be less than 10 seconds apart. Take as many time points as you can so that you have the best possible data set for measuring the initial rate.

The substrate concentrations should range from about 5 times higher than the Km to about one-fifth the Km. The inhibitor concentrations should range from about 5 times the IC50 to about one-fifth the IC50.

@ Adam B Shapiro 

How can I calculate velocity of reaction from absorbance of various concentration of substrate (with or without sample) which need to be plotted with substrate concentration to specify the inhibition mechanism for inhibitors.

In an absorbance-based assay in which the product and substrate have different extinction coefficients, the change in absorbance is proportional to the amount of product formed. Therefore the slope of the initial linear part of the absorbance versus time curve is the rate of product formation. Plot this slope versus the substrate concentration to prepare a Michaelis plot for each inhibitor concentration.

March 25/016

Dear Muhammad

Adam Shapiro presented you a useful generality regarding your expected experiments with the alpha-glucosidase inhibition. In all cases, initial rate activity (initial velocity: V0) must be respected. V0 is function of the E concentration and the time unit (5,10, 20 min, etc). 4 reading are at least required to plot a linear curve, i.e. activity versus time. Consider the linear part of each plot: V0 is the slope (derivative of the linear curve). Since you repeat the enzyme assay several times in presence or absence of inhibitor (I guess this is a flavonoid compound) as substrate concentration function, several values of V0 and V0i (i for inhibitor) are expected. Calculate 1/V0 and 1/S (reciprocal values) and plot them to get linear curves according to Linweaver-Burk plot. Then, determine the inhibition type (I expect it is non competitive). You can consider the values of V0, V0i in their term of absorbance (e.g., 0.5 per 20 min; this must be proportional to the considerd time and the enzyme amount). Substrate concentration must be expressed in M or mM (i.e., mol or mmol per L of the assay medium). V0, V0i can be expressed as microM (micro mol per L of the assay medium) per min (see the attached equation) or  microM per the assay time, or microM per min per mg enzyme (or protein).

I hope this explanation is helpful.

The incubation time of my END POINT assay was 40 minutes so I should keep the same time as overall period for reaction completion? 

Not at all. Initial velocity is an activity, proportional to the E amount during the applied time. Besides and in completion to the equation already transmitted, you have to consider the well deep, if it is for example  0.5 cm, then you divide the equation on this value.

The length of time from the start in which the velocity is constant (that is, the progress curve is linear) will vary according to the substrate concentration, with higher substrate concentrations resulting in longer periods of linearity. Also, the period of linearity will decrease if the enzyme concentration decreases. You can adjust the enzyme concentration downwards if needed to get more data points during the initial velocity period. To gain some experience with these factors, try running the reaction at several substrate concentrations and enzyme concentrations and measuring how long the reaction progress curve remains linear under each set of conditions.

Salam all; I have not an answer, but a question in the context

I want to try the anti-invertase activity of plant extracts.So, I want to purchase the enzyme, I must choose between two offers: Invertase from baker's yeast ≥300 units/mg solid with storage temp. −20°C and  Invertase from baker's yeas 200-300 units/mg solid with storage temp. 2-8°C. For the first offer, the storage temperature (-20°C) is difficult to put on for the long time that I need, the enzyme may die at higher temp. In the other hand, the second enzyme is of 200-300 units/mg solid, so, with this range I can't calculate the concentration for the assay in U /ml. What I must choose, I want the second, but how can I solve this problem...

Thanks so much for yours answers.

The actual measurement of U/mg in the second product will probably be available, either printed on the label or on-line. The range given, 200-300 U/mg, usually means that the actual measurement will fall in between those values. The same goes for the >300 U/mg product, which is presumably of higher purity than the 200-300 U/mg product. The 200-300 U/mg product may contain excipients, such as sugar, to help maintain its stability during storage at 2-8oC, which are not present in the purer product.

Adam B Shapiro

Thanks so much for the answer. In fact, I had tried hard to find the actual measurement of the product, which is available in Sigma Aldrich page...But I didn't find any additional information.

Dear all, I am studying the inhibition of α-Glucosidase using two inhibitor compounds. In the experiment, I find that the two compounds exhibit synergistic inhibition. Unfortunately, I don’t know how to explain the mechanism of synergistic inhibition. I search some reference papers, however, very few papers mention the mechanism. I suffer from this problem couple of days. If possible, could you please give me some suggestions about determining the mechanism? Thanks very much!

You must consider multiple possibilities:

(1) One or both of the inhibitors interferes with the readout of the assay. This is tested by starting with the detected product of the reaction and seeing whether the inhibitor inhibits its detection. This is the simplest test. Start here.

(2) One or both of the inhibitors is non-specific. This is very common. The binding stoichiometry is greater than one for such an inhibitor. It may act by causing denaturation of the enzyme, or it may form a soluble aggregate to which the enzyme binds and loses activity. Is the inhibition time-dependent? Is the inhibition only partially reversible? Is the Hill slope of the IC50 curve greater than 1? Does the inhibitor lose potency when you increase the enzyme concentration in the assay and shorten the reaction time? Does the inhibitor lose potency when you include 0.01% Triton X-100 detergent in the reaction? These are all signs of non-specific inhibition.

(3) Both inhibitors are specific, stoichiometric binders of α-Glucosidase. Two inhibitors will always show more inhibition than one inhibitor, even if they can't both bind at the same time. For 2 inhibitors to show greater than additive inhibition, they must bind simultaneously to separate places on the enzyme. Study each inhibitor separately. Are they competitive with the substrate, non-competitive, or uncompetitive? Measure the binding stoichiometries and affinities of the inhibitors if you can.