**5. Biophysical techniques to determine MOA**

Various biophysical methods can be used to explain the MOA of AMPs. These methods depend upon bacterial components (e.g., DNA, lipid extracts, nucleotides, etc.). They provide significant insights on the MOA in-depth specifically when the MOA does not include membrane disruption.

### **5.1 Membrane disruption**

The membrane damaging MOA has been the focal point of many researchers for many years [6]. Following are the techniques to determine membrane disrupting mechanisms.

### *5.1.1 Pyranine leakage assay*

Pyranine (Trisodium 8-hydroxypyrene-1,3,6-trisulfonate) is a pH-sensitive hydrophilic polyanionic molecule used as a fluorescent dye to detect the quantity of internal aqueous proton in phospholipid vesicles. Pyranine and anionic phospholipid vesicles have no considerable interaction, due to the anionic nature of pyranine. These properties utilize the use of pyranine molecules to examine the transport of hydrogen ions and counterion across phospholipid vesicle membranes even in the presence of AMPs that disrupt membranes [56, 57]. The accessory information provided through this method is that leakage is affected by membrane composition or ions which are very crucial for activity, like Ca2+ [2].

### *5.1.2 Calcein leakage assay*

Calcein leakage assay can be used to study the ability of AMPs to disrupt the lipid bilayers like large unilamellar vesicles (LUVs) or small unilamellar vesicles (SUVs). Calcein or carboxyfluorescein is an aqueous soluble fluorescent dye that is entrapped into LUVs and a gel filtration mechanism is used to eliminate the non-entrapped calcein after self-quenching at critical extreme concentrations [7]. If peptides cause membrane disruption or create large pores in the bilayer, this can lead to leakage of entrapped calcein out of the vesicle lumen thus relieving the selfquenching and resulting in an increase in fluorescence emission intensity [58]. This assay can also be utilized to understand the effect of AMP on bacterial cytoplasmic membrane integrity [59].

#### **5.2 Membrane interaction**

Following techniques are used to explain the mechanism of how antimicrobial peptides interact with the plasma membrane.

#### *5.2.1 Oriented circular dichroism*

It is significantly crucial to understand the interaction of AMPs with model membranes since many AMPs show their activity by traversing through the

#### *Antimicrobial Peptides: Mechanism of Action DOI: http://dx.doi.org/10.5772/intechopen.99190*

membranes. Oriented circular dichroism (OCD) is the method by which we can study the interaction. Oriented lipid bilayers are employed in OCD to gain insights into the peptide membrane alignment [7]. A clear difference between parallel vs. perpendicular localization of a peptide concerning the bilayer membrane can be observed through the signal [60]. This method is mostly studied on α-helical peptides. A change in the CD signal is observed when peptides form well-defined pores with respect to increase in peptide concentration [7].
