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Aggregation on Target Proteins: P.abrotanoides Karel Role

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Published: Tue, 08 May 2018

There is direct relation between proteins structure and their function. Hydrophobic areas has a less contact with water. In contrast, hydrophilic areas absorb with water. With change in the biochemical compounds of humans body such as PH, temperature or bodys chemical condition in protein structure change as well and these changes can cause denaturation of protein.

Some diseases can let of significant change in the protein structure. In this cases the hydrophobic areas of proteins fuse together and make heavy structure which aggregate in intra- or extracellular. Disorder (amorphous) and order (amyloid fibril ) are types of aggregation, their formation is dependent on environmental condition such as temperature, PH, and reducing agent.

There are many diseases with shown protein aggregation such as Alzheimer and Parkinson. For example in Alzheimer, proteins aggregated in brain.

In our study, target proteins (ovotransferrin, α-lactalbumin and insulin) have disulfide bonds. Protein (ovotransferrin, α-lactalbumin and insulin) can be denatured and aggregated in vitro by adding DTT (reducing agent). Researchers have been using of them in vitro for finds materials that prevention protein aggregation.

In the current days people use herbs as part of their diets or medicine for treatment diseases. One of this herb is P.abrotanoides karel to lamiceae family. If can be find in Iran, Pakistan and Turkmenistan. People use it as Antibiotic against bacterial and viral diseases in addition it works as Anti-inflammation medicine. P.abrotanoides karel has wide range of use full effect on diseases. (herbal medicine have less destructive effect on patient in the comparison with synthetic drugs.)

In this study, we investigate the role of P.abrotanoides karel in aggregation on target proteins (ovotransferrin, α-lactalbumin and insulin). The result shows that P.abrotanoides karel extract inhibit protein aggregation. There was direct relation between protein aggregation prohibition and P.abrotanoides karel concentration.

All target proteins have disulfide bonds in the structure, they aggregated after addition of DTT and reduction of their disulfide bonds.

In this study, the ability of P.abrotanoides extract to prevent DTT-induced aggregation on all target proteins was measured by light scattering absorption.

In the absence P.abrotanoides, all proteins aggregated about 30 min after the addition of DTT (fig 1. a, b and c).pp

Ovotransferrin aggregation decreased by ….% at 1:0.25 w/w of ovotransferrin : P.abrotanoides extract. ovotransferrin aggregation with P.abrotanoides concentration, sach that at 1 : 2 w / w ratio of ovotransferrin : P.abrotanoides extract protective effect increased by ….% (fig 1.a and table 1).

P.abrotanoides extract protective of α-lactolbumin, shown in fig 1.b at 1 : 0.25 ratio of α-lactolbumin : P.abrotanoides extract, protective effect was …% but the reduction in ratio 1 : 2 w / w α-lactolbumin : P.abrotanoides extract was …%. The reduction of α-lactolbumin aggregation was increased by increasing the concentration of P.abrotanoides extract (fig 1.b and table 1).

According to these data (fig 1.c ), insulin alone showed high light scattering but in the presence P.abrotanoides extract, insulin aggregation decreased, by increasing the extract, insulin aggregation was decreased such that in 1 : 2 w /w insulin : P.abrotanoides extract was about …..%.

51.29

Ovo : p (1 :0.25)

78.57

Ovo : p (1 : 0.5 )

98.70

Ovo : p (1 : 1 )

100

Ovo : p (1 : 2 )

37.66

α-la : p (1 : 0.25

41.38

α-la : p (1 : 0.5 )

50.89

α-la : p (1 : 1)

100

α-la : p (1 : 2 )

21.48

Ins : p (1 : 0.25)

27.02

Ins : p (1 : 0.5)

50

Ins : p (1 : 1)

84.59

Ins : p (1 : 2)

Tryptophan (trp) has intrinsic fluorescence, changing trp situation causes changing intrinsic fluorescence intensity in the target proteins. There is direct relation between protein’s structure and intrinsic fluorescence intensity, protein’s conformation change less while fluorescence decreasing. P.abrotanoides extract alone as high concentration shows very low intrinsic fluorescence intensity ( approximately zero).

We were measured change in protein structure by intrinsic fluorescence intensity when target proteins were denatured and aggregated by DTT.

There isn’t trp in insulin’s structure, therefore we didn’t studied intrinsic fluorescence intensity in it.

In the absence P.abrotanoides extract, ovotransferrin’s intrinsic fluorescence intensity increased after addition of DTT, therefore ovotransferrin’s structure was changed by DTT (fig 2.a). In the presence P.abrotanoides extract, we found decreasing in ovotranferrin’s fluorescence, then P.abrotanoides extract prevented of denaturation agent (DTT). Intrinsic fluorescence intensity decreased about …% in the ratio of 1 : 0.25 w /w ovotransferrin : P.abrotanoides extract, this amount increased to …% at 1 : 2 w /w ovotransferrin : P.abrotanoides extract.

α-lactalbumin structure in the absence and presence of P.abrotanoides extract was evaluated by intrinsic fluorescence intensity. In the absence P.abrotanoides extract, α-lactalbumin’s fluorescence was increased, according to these data, α-lactalbumin’s conformation was changed by DTT (fig 2.b). In the presence P.abrotanoides extract α-lactalbumin’s fluorescence decreased, therefore it conformation changed less. α-lactalbumin was denatured less by increasing the concentration of the P.abrotanoides extract. For example at 1 : 0.25 w/w ratio of α-lactalbumin : P.abrotanoides extract, fluorescence intensity decreased about …% but at 1 : 2 w/w of α-lactalbumin : P.abrotanoides extract, fluorescence intensity was …%.

ANS is an external fluorescence, it connect to hydrophobicity of target proteins then created fluorescence, therefore ANS binding assays have shown changing in protein structure.

In the results shown, in the absence P.abrotanoides extract, ANS fluorescence was increased by DTT, therefore protein’s hydrophobic areas was changed and proteins was denatured by DTT.

In the next experiment, ANS binding was measured in all target proteins in the presence of P.abrotanoides extract. extract decreased extrinsic fluorescence intensity, then P.abrotanoides extract prevented as changing in proteins conformation.

The secondary structure was studied by Fur-UV (190-250 nm) CD spectroscopy in the absence or presence P.abrotanoides extract.

We were monitored the α-lactalbumin secondary structure by Fur-UV (190-250 nm) CD spectroscopy. In the absence P.abrotanoides extract, DTT was deleted positive peak at 192 nm and negative peak at 208 nm, therefore it was created β-sheet and disappeared α-helical in the α-lactalbumin structure. In contrast, in the presence P.abrotanoides extract, we found one positive peak at 192 nm, spectrum hasn’t positive peak at 195 nm. This results shown, extract prevented of creating β-sheet and it protected of α-helical (Fig 4.a).

Fig 4.b is Far-UV spectrum of insulin. In the presence P.abrotanoides extract, spectrum showed two negative ellipticity at 208 nm 222 nm and one positive ellipticity at 192 nm and. According to these data, P.abrotanoides extract protected of α-helical structure in insulin, but in the P.abrotanoides extract, Far-UV spectrum showed positive peak at 195 nm and negative peak at 180 nm, therefore β-sheet was increased by DTT.

Many of the proteins have been aggregated by environment agents like unfavorable PH, temperature and reducing agents. Proteins were creating many diseases in the humans When they aggregated in their tissues, then it is important that we studied on proteins aggregation.

Chaperones are protective molecules of body’s proteins, they have helped to folding process in proteins and prevented of their aggregation, therefore they have more useful function in prevention of many diseases like Alzheimer and diabetes type II.

Many of chaperone’s functions have been found in vitro. Because researchers have studied on various materials in vitro that they may prevention of protein aggregation. For the example, ghahghaei et al found Crocin protective effect on aggregation of Aβ1-40.

P.abrotanoides is a medicinal natural plant. It is used as a refrigerant, anti-inflammatory, infection, anti-bacterial and pain-killer, it has antioxidant effect. It is growing wild in Iran, Pakistan, Afghanistan and Turkmenistan. We guessed P.abrotanoides may prevent of protein aggregation because it has many useful effect. Therefore we investigated P.abrotanoides effect on protein aggregation in vitro.

Light scattering is a method that it reveals aggregation of target protein. Different sized proteins were aggregated by DTT in the absence or presence P.abrotanoides. P.abrotanoides was good inhibitor for protein aggregation. Furthermore protein aggregation was decreased by increasing of P.abrotanoides concentration.

We guess P.abrotanoides was prevented of protein aggregation by interaction with hydrophobic areas of target proteins or interaction with DTT.

Intrinsic fluorescence determine the changing of trp position and conformation in the target proteins (ovotransferrin and α-lactalbumin). In the second method, the changing in protein’s conformation was measured by the intrinsic fluorescence in the absence or presence P.abrotanoides extract. As shown in fig 2, P.abrotanoides decreased intrinsic fluorescence intensity, therefore it prevent of changing trp position in proteins and creating protein aggregation.

ANS is good method for monitoring hydrophobic areas in the proteins (ovotransferrin, α-lactalbumin and insulin). Fluorescence intensity has been increased by connection ANS with hydrophobic areas when DTT increased the exposure of the hydrophobic residues. Our results indicated that P.abrotanoides extract can prevent the expose of the hydrophobic residues and finally prevent protein aggregation.

Secondary structure of proteins is determined by CD spectroscopy. We can say that CD spectroscopy is a method for confirming pervious results. In the far-UV CD spectroscopy data, we observed that α-helical was decreased by DTT, but β-sheet decreased. In contrast P.abrotanoides exract increased the helical secondary structure of the all target proteins and decreased β-sheet, therefore extract has protective effect on protein structure,then it caused decreasing on the protein aggregation.

Stresses causes denaturation and exposes the hydrophobic regions of proteins. The investigations showed that P.abrotanoides has terpinolene. We guesses, terpinolene can be connect with hydrophobic areas of all target proteins and they are prevented of formation heavy structures and their aggregation.

In the other theory, extract may be connected with DTT and it prevent from the reaction of DTT with the target proteins.

In conclusion we can say that P.abrotanoides can be inhibit of aggregation’s diseases or it can use for treating aggregation’s diseases. But we suggest researcher can be find effective materials of P.abrotanoides extract in other searches, then they will be testing it materials on animal’s models. It is hoped that P.abrotanoides extract will treat of aggregation diseases or will inhibit of their.


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