1. Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran 3. Department of Biochemistry and Biophysics, faculty of sciences, Mashhad Branch, Islamic Azad
Abstract
Introduction
Caseins are the dominant phosphoproteins of mammalian milk and they can act as micelles that are made of polypeptides known as α, β and κ-caseins. α-casein contains two tryptophan(trp) while β and κ-caseins have one tryptophan residue. α-caseins are the major casein proteins containing 8-10 seryl phosphate groups. mammalian milks contain large quantities of protein -based particles (the casein micelles). they note that the phosphoseryl clusters in α-s1 and b-caseins are highly conserved and that despite high rates of mutational change and sequence diversity, many of the mutational also tend to be conservative with hydrophobic residue.
ostrich egg white(oew) proteins were hydrolyzed by trypsin to identify inhibitory peptides of angiotensin i-converting enzyme (ace). angiotensin converting enzyme(ace) is an important enzyme in renin-angiotensin system which increases blood pressure by catalyzing the conversion of angiotensin ii is of great importance.
Methods
α-casein and kh2po4 were purchased from sigma-aldrich co. (st louis, mo. usa). the ace peptide was extracted from ostrich egg white (oew) proteins. α-casein was dissolved in potassium phosphate buffer solution (50 ml, ph= 7.4) at concentration of 1.3*10-2 mm and the injection of ace inhibitory peptide at concentration 0.5 mm done in per 3minutes for 40 times.
Results
To investigate the conformational changes of α-casein structure due to binding to ace inhibitory peptide, we used zeta potential spectroscopy. the negative values of zeta potential show the domination of electrostatic interactions. as the diagram shows in fig.1 zeta potential value decrease by increasing the concentration to ace inhibitory peptide. the amounts of zeta potential measurments show that the dominant force in interaction of α-casein-peptide is hydrophobic and the polarity of the interaction is low. as the diagrams show in fig. 2.a and fig. 2.b, fluorescence intensity of the protein decreases by increasing the concentration of ace inhibitory peptide. this process is named concenration-dependant quenching. further more, there was a slight blue shift at the maximum wavelenght of α-casein (from 340nm to 337nm). after the addition of ace inhibitory peptide, it suggests that the microenviroment of tryptophan residues was changed after the addition of ace inhibitory peptide. this transition occurs when trp and tyr aminoacids transfer to hydrophilic enviroment and it demonstrates the decrement of the polarity of these two chromophores. the occured quenching in fluorescence spectrum of this protein in presence of ace peptide demonstrates the formation protein-ligand complex. the quenching value in 280nm excitation wavelenght in more than quenching value in 295nm excitation wavelenght because 280nm wavelenght is related to excitation of electrons deployed in electron layers of tyr and trp amino acids, while 295nm wavelenght only caused trp the electronic excitation.
Conclusion
In this article we investigated the interaction between α-casein and ace peptide at standard conditions by using fluorescence and zeta potential spectroscopy. according to the anti oxidant effects of ace inhibitory peptide and its ability of blood pressure decrement, this peptide can also be used due to treatment of cancer and cardiovasculare deseases.
as casein protein is an important phosphoprotein with anti cancer effects and it has many application in food-processing industries, the possibility of interaction between ace inhibitory peptide with α-casein protein was evaluated.
the results fluorescence spectroscopy studies show that the presence of ace inhibitory peptide causes the quenching of emission of protein fluorophores. a little blue shift shows that the environment of around of fluorophores has been more polar and these yield transitions are caused by the formation of ligand-protein complex.
the results of zeta potential spectroscopy measurments have shown that the main forces in that interaction are hydrophobic forces and the electrostatic forces are increased by the changes of the amounts of concentration.
