Solvation, Thermodynamic and UV-Visible Studies of Non-Aqueous Peptide Solutions

Solvation, Thermodynamic and UV-Visible Studies of Non-Aqueous Peptide Solutions

Dr. R. Padmavathy

Head & Associate Professor,

PG & Research Department of Physics, Seethalakshmi Ramaswami College, Tiruchirappalli- 620002,

Tamil Nadu, India.

N. Radha

K. Dhanalakshmi

Research Scholar,

PG & Research Department of Physics, Seethalakshmi Ramaswami College, Tiruchirappalli- 620002,

Tamil Nadu, India.

Associate Professor,

PG & Research Department of Chemistry, Seethalakshmi Ramaswami College, Tiruchirappalli- 620002, Tamil Nadu, India.

Abstract – Peptides are most important molecules due to their wide application in drug production and their role as signal transmitters in the cell communications. The systematic study of peptides can prove valuable information about their behaviour in solution and insight into the conformational stability of proteins. Amino acids and peptides are the fundamental structural units of protein. The properties of protein such as their structure, solubility, denaturation activity of enzymes, etc. are generally influenced by electrolytes. The study of intermolecular interaction plays an important role in the development of molecular sciences. Large number of studies have been made on the molecular interaction in liquid systems by various physical methods like ultra violet and ultrasonic technique. The present investigation deals with the study of molecular interaction of non-aqueous dipeptide solution of various concentrations and at different temperatures. Using the experimental data, solvation number (Sn) and internal pressure (i) are computed by using the standard formulae. These parameters have been interpreted in terms of solute-solvent interaction in the solutions. The results obtained from thermodynamic, UV – visible spectroscopic and antimicrobial study reveals the entry of solute into the peptide solution.

Keywords: Ultrasonic velocity, internal pressure, UV Visible spectra, E.Coli

I.INTRODUCTION

Ultrasonic is a versatile non-destructive technique and highly useful for the investigation of various physical properties such as residual stress, hardness, grain size micro structure, elastic constant etc. Recent developments have found use of ultrasonic energy in medicine, engineering and agriculture. Ultrasonic study on the amino acids with aqueous solution of electrolytes and non- electrolytes provides useful information in understanding the behavior of liquid systems1. The ultrasonic study of liquid is very important in understanding the nature and strength of molecular interactions. The biological activity of drug molecules and the activation energy of the

metabolic process 2 basically depend on the type and strength of the intermolecular interactions. Ultrasonic velocity studies of amino acids 3-4, peptides 5-9 and proteins 10 in aqueous media, aqueous urea solution, mixed aqueous solutions and organic solvents have been carried out by number of researchers for investigation of solute- solute, solute-solvent intermolecular/ interionic interactions. However few authors have studied the behaviour of amino acids and peptides in aqueous electrolyte solutions. The Glycyl-L-glycine+ sodium nitrite+ formamide system into gives the interesting information about various interactions operative in solution. The Spectroscopic techniques are used to determine the structure and function of bio- molecules. The results arrived from the internal pressure and solvation number parameters were correlated with UV- visible and antimicrobial study.

1. MATERIALS AND METHODS

   Ultrasonic velocity was measured using Digital ultrasonic interferometer of fixed frequency 2MHz (Model F-81 Mittal Enterprises, New Delhi) with an accuracy of ± 0.2m/s. The density of the non aqueous solutions of peptide with electrolyte are measured using Anton Paar DMA 4100 Digital Densitometer with an accuracy of ± 0.0001 gm/cc. The viscosity of solutions are measured using Cannon Fenske Viscometer (± 0.1 %) with the experimental solutions immersed in a temperature controlled water bath. The time of flow was measured using a stop watch with an accuracy of 0.1 sec. The derived parameters such as solvation number (Sn) and internal pressure (i) are calculated by the formulae given below

   Solvation number Sn= ni/nf [1- soln/ solv]

   Internal pressure

   eff

   i= bRT [K/u]1/2 [2/3/M 7/6] (109 Pa)

2. RESULTS AND DISCUSSION

   1. Solvation number

      The solvation number is computed from compressibility measurements. The solvation number is positive at all temperatures and at higher molalities. The solvation approach is used to interpret ion-solvent interaction. The decrease in solvation number with increasing molality is due to either not enough solvent molecules available for the ions or preferentially ion- pairing occurred. In very dilute solution, there is a basic structural changes happens in the first co-ordination sphere. Hence the filling of cation and anion influence spheres occurred in a set of sequence in relation to other solvation. The solvation number of an ion depends on the solvent.

   2. Internal pressure

   Internal pressure represents the resultant of the forces of attraction and repulsion between the molecules. In the present investigation, the i increases with increasing concentration. When a non-aqueous electrolyte solution is added to Glycyl-L-glycine dipeptide, it attracts the more solvent molecules towards itself by wrenching the molecules

   2 3

   When the temperature is increased, there is a tendency for molecules to move away from each other, reducing the cohesion between them. Thus i decreases as it is a measure of cohesive energy. The dipeptide tightens the formamide molecules around it, hence the molecules comes closer together. In the present dipeptide systems, this may be taking place as solvophilic group interactions between the ions of sodium nitrite (Na+, NO -) and (NH +, COO-), (-OH) group of peptides . (i.e) Solvophilic group interaction between the ions of sodium nitrite and non- polar parts of peptides.

   Table: 1 The computed values of solvation number and Internal pressure of non aqueous ternary solutions at different temperatures are zone in the table

   Fig 1: Solvation Number

   Fig 2: Internal Pressure

   SAMPLES	200-400 nm
   Formamide (Solvent)	233.03
   Glycyl-L-glycine (solute-I)	293.22 280.93
   Sodium-Nitrite (solute-II)	234.62
   0.001m solution of Glycyl-L- Glycine with 0.01m of Sodium nitrite in Formamide	334.34
   0.01m solution of Glycyl-L- Glycine with 0.01m of Sodium nitrite in Formamide	323.01 339.83
   Saturated Solution of Glycyl-L- Glycine with Sodium nitrite in Formamide	317.43 350.04

   Table: 2 UV VISIBLE SPECYTRA OF THE SAMPLES

   Molality (m)	Solvation Number			Internal Pressure (109Pa)
   	308.15 K	318.15 K	328.15 K	308.15 K	318.15 K	328.15 K
   0.001	9.34	22.81	17.85	1.3135	1.1912	1.1028
   0.005	14.56	18.82	21.21	1.3295	1.2196	1.1107
   0.1	13.96	14.78	18.84	1.3539	1.2236	1.1148
   0.025	10.55	9.41	13.55	1.3570	1.2362	1.1312
   0.05	7.55	6.14	9.08	1.3749	1.2462	1.1407

3. UV-VISIBLE SPECTRUM ANALYSIS

   ACIC

   St.Jo sep h' s Colle ge, T rich y-2

   Glycyl-L-glycine exhibit two peaks at 293 and 280 nm. The UV-Visible spectra of sodium nitrite exhibits a sharp peak at 334.6nm which may be due to intermolecular interactions in the solid state of sodium nitrite. At 0.001m molality a single peak is found at

   3.20

   3.0

   2.8

   2.6

   2.4

   2.2

   2.0

   1.8

   1.6

   A

   1.4

   1.2

   1.0

   0.8

   0.6

   0.4

   Spe ctru m Na me: SRC-FORM.SP

   2 3 3 .0 3 ,2 .51 6 9

   Date: 7/1 /2 01 5 UV-Vis Spe ctrum

   334.3nm.At 0.01m molality two peaks are observed at 323nm and 339.8nm.On dissolution of Glycyl-L-glycine in sodium nitrite formamide mixture at the UV-Visible spectral peaks are found at 317.4nm and 350nm of high intensity (~3.18-3.24). The interaction between solute and

   solvent is represented as Figure 3 respectively. The sodium

   0.2

   -0. 01

   190.0 300 400 500 600 700 800 900 1000 1100.0

   nm

   Instrument Model: Lambda 35 Sc an Speed: 480.00 nm/min Data Interval: 1.0000 nm

   Fig 4:UV-Vis Spectrum of Formamide

   ACIC

   St.Jo sep h' s Colle ge, T rich y-2

   nitrate peak at 334.6nm has shifted 17.6nm hypsochromically and 5.4nm bathochromically and solute- solvent interaction is represented as in Ia+Ib. The peak at 317nm has bathochromically shifted from 317-323nm by 6nm, but the peak at 350nm has shifted hypsochromically

   0.100

   0.09

   0.08

   0.07

   0.06

   0.05

   0.04

   0.03

   A 0.02

   0.01

   0.00

   -0. 01

   Spe ctru m Na me: GG-Sa lt-.SP

   293.22,0.013269

   280.93,-0.012032

   Date: 7/1 /2 01 5 UV-Vis Spe ctrum

   from 350-339.8nm by 11.2nm and the solute-solvent interaction.

   The peaks at 823nm + 339.8nm have merged at 334.3nm. There is a bathochromic shift of 11.3nm + 5.5nm of both peaks and correspond to III. The above results indicate

   -0. 02

   -0. 03

   -0. 04

   -0. 050

   280.0 400 500 600 700 800 900 1000 1100.0

   nm

   Instrument Model: Arithmetic Sc an Speed: 480.00 nm/min Data Interval: 1.0000 nm

   Fig 5:UV-Vis Spectrum of Glycyl-L- glycine

   ACIC

   St.Jo sep h' s Colle ge, T rich y-2

   compact structure up to 0.01m and good solute-solvent interaction at 0.001m.

   Spe ctru m Na me: SD-Salt-.SP

   0.0500

   0.045

   0.040

   0.035

   Date: 7/1 /2 01 5

   UV-Vis Spe ctrum

   Fig 3: Solvated Structure of Glycyl-L-Glycine + Sodium Nitrite + Formamide

   0.030

   0.025

   0.020

   0.015

   0.010

   A

   0.005

   0.000

   -0. 005

   334.62,0.018513

   -0. 010

   -0. 015

   -0. 020

   590.75,-0.023036

   755.06,-0.017972

   985.95,-0.017428

   KETO FORM

   -0. 025

   -0. 0300

   300.0 400 500 600 700 800 900 1000 1100.0

   nm

   H O

   Instrument Model: Arithmetic Sc an Speed: 480.00 nm/min Data Interval: 1.0000 nm

   Fig 6:UV-Vis Spectrum of Sodium Nitrite

   C

   H

   H N H H

   O N

   2.20

   2.0

   1.8

   ACIC

   St.Jo sep h' s Colle ge, T rich y-2

   Spe ctru m Na me: GG-S-00 1-.SP

   334.34,1.5169

   Date: 7/1 /2 01 5 UV-Vis Spe ctrum

   O C

   CH2

   NH C

   O H O N O CH2

   Na

   1.6

   1.4

   1.2

   A

   1.0

   0.8

   0.6

   0.4

   0.2

   Keto form of formamide

   -0. 03

   260.0 300 350 400 450 500 550 600 650 700 750 800 850 900.0

   nm

   H

   C OH

   Instrument Model: Arithmetic Sc an Speed: 480.00 nm/min Data Interval: 1.0000 nm

   Fig 7: UV-Vis Spectrum of of 0.001m

   ACIC

   St.Jo sep h' s Colle ge, T rich y-2

   O

   H N H H

   3.50

   3.4

   3.2

   3.0

   Spe ctru m Na me: GG-S-01 -.SP

   323.01,2.9380

   339.83,2.8938

   Date: 7/1 /2 01 5 UV-Vis Spe ctrum

   2.8

   2.6

   2.4

   2.2

   O C O

   NH

   CH2 C

   N CH2

   H O N O

   Na

   2.0

   1.8

   A

   1.6

   1.4

   1.2

   1.0

   0.8

   0.6

   0.4

   0.2

   -0. 04

   260.0 300 350 400 450 500 550 600 650 700 750 800 850 900.0

   nm

   Instrument Model: Arithmetic Sc an Speed: 480.00 nm/min Data Interval: 1.0000 nm

   Fig 8:UV-Vis Spectrum of 0.01m

   Enol form of formamide

   3.80

   3.6

   3.4

   ACIC

   St.Jo sep h' s Colle ge, T rich y-2

   Spe ctru m Na me: GG-S-Sat-.SP

   317.43,3.2443

   350.04,3.1840

   Date: 7/1 /2 01 5 UV-Vis Spe ctrum

   incubated at room temperature for 2 to 4 days. During this period the drug diffuse through the agar and inhibit the growth if the drug is potent. The diameter of inhibiting

   3.2

   3.0

   2.8

   2.6

   2.4

   2.2

   2.0

   A 1.8

   1.6

   1.4

   1.2

   1.0

   0.8

   0.6

   0.4

   0.2

   -0. 03

   260.0 300 350 400 450 500 550 600 650 700 750 800 850 900.0

   nm

   zone around the disc was measured by using the ruler and Interpretation has to be assessed based on the following table.

   Instrument Model: Arithmetic Sc an Speed: 480.00 nm/min Data Interval: 1.0000 nm

   Fig 9:UV-Vis Spectrum of Saturated Solution

4. SALIENT FEATURES OF THE SAMPLES TEST ORGANISMS:

   The test microorganisms of E.Coli and Fungus Aspergillusniger, are obtained from National Chemical Laboratory (NCL) Pune. Antibacterial and antifungal properties of the ligand and its complexes were tested in vitro against the bacterial species Escherichia coli; fungal species Aspergillusniger, by the disc diffusion method.

   A.Antibacterial activity

   The NCIM numbered strains bought from National Chemical Laboratory (NCL) Pune was periodically Sub cultured in Nutrient agar and maintained in the laboratory. The Strains namely Proteus vulgaris and Klebsiellaaerogenes were brought to the active phase by sub culturing in Nutrient broth and incubated at 37°C for

   18 hours.The Standardized inoculam about 0.1 ml was inoculated on Muller hinton agar (Hi media) Uniformly. The sterile disc (watt man No.2 of 6mm diameter was placed at equal interval on uniformly inoculated plate and a standard disc Ciprofloxacin 5 mcg/disc was also placed by aseptic technique). The plates were incubated at 37°C for 24 hours. During this period the drug diffuse through the agar and inhibit the growth if the drug is potent. The diameter of inhibiting zone around the disc was measured by using the ruler and Interpretation has to be assessed based on the following table.

   B. Antifungal activity

   The ATCC numbered strains bought from Madras Medical College was periodically sub cultured in Sabouraud dextrose agar and maintained in the laboratory. The strains namely Aspergillusniger, Aspergillusflavus, Aspergillusfumigatus, Mucor& candida albicans were brought to the active phase by sub culturing in Sabouraud dextrose broth (table 2) and incubated at room temperature for 4 days. The standardized inoculam about 0.1 ml was inoculated on Sabouraud dextrose agar uniformly. The sterile disc watt man No.2 of 6 mm diameter was placed at equal interval on uniformly inoculated plate and a standard disc Nystain 100 units/disc was also placed by aseptic technique. The test sample about 100 µl was loaded to the sterile disc by using aseptic precautions. The plates were

   S.No	Zone of inhibition in mm	Report
   1.	Zone of inhibition between 6mm to 12mm	Intermediate
   2.	Zone of inhibition below 6mm	Resistent
   3.	Zone of inhibition more 12mm	Sensitive

   Report on Antimicrobial activity of the given samples

   The anti-microbial activity for the given Sample was carried out by disc diffusion Technique (Indian Pharmacopoeia 1996, Vol II a-105). The test microorganisms from National Chemical Laboratory (NCL) Pune and maintained by periodical sub culturing on Nutrient agar and Sabouraud dextrose agar medium for bacteria and fungi respectively. The effect produced by the sample was compared with the effect produced by the positive control (Reference standard Ciprofloxacin 5

   µg/disc for bacteria; Nystatin 100 µg/disc for fungi).

   S. N o	Name of the Microorganis ms	Zone of inhibition in mm
   		G G	So d.N it	GG+ Sod.Ni t	FMA (Solvent)	Standar d
   1.	E.coli (NCIM 2065)	16	18	20	08	38
   2.	Aspergillus niger (NCIM 105)	12	17	16	10	30

   In the present investigation, it is identified that the non- aqueous solutions of Glycyl-L-glycine with Sodium Nitrite act as an intermediate with respect to E.coli whereas this combination is sensitive in the case of Aspergillus niger, ie this combination may act as an disinfectant.

   Antibacterial activity (E.Coli)

   Antifungal activity (Aspergillus niger)

   Fig 10: Antimicrobial activity of Glycyl L glycine + Sodium Nitrite + Formamide solutions

5. CONCLUSION

   Analysis of the transport properties suggest that there is a strong solute-solvent interaction exist in the ternary solution. The above results are confirmed by solvation studies. UV-Visible spectra are reveals that there is a strong interaction takes place in ternary solutions. The (N-C-O) peptide linkage is dependent on the structure flexibility of the molecule involved and is of importance in the synthesis of protein. An attempt is made to corroborate with solvation, thermodynamic and spectroscopic analysis.

6. ACKNOWLEDGEMENT

   The authors are thankful to UGC for the financial support towards Major Research Project 42-858 (SR (20132017)), ACIC Instrumentation center, St. Josephs college, for recording the UV-Visible spectra and Periyar College of Pharmaceutical Science, Tiruchirappalli, Tamil Nadu, India for the antimicrobial studies.

7. REFERENCES

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2. Palaniappan, L and Karthikeyan, V., (2005) Ultrasonic analysis in the ternary mixtures of 1,4 diaxane+ carbon tetrachloride +1-butonal Ind. Journal of Phy.79 (2),153-156.

3. Rodriguez, H., Soto, A., Arce, Khoshkborchi.,(2003) Apparent molar volume isentropic compressibilities refractive index and viscosity of Dl-Alanine in aqueous NaCl solutions J. Solution Chem. 32. 53-63.

4. Banipal, T.S., Singh, G., (2004) Partial molaradiabatic compressibilities and viscosities of some amino acids in aqueous 1,4 dioxane solutions at 298.15K Indian J. Chem. 43 A.1156-1166.

5. Banipal, T.S., Sehgal, G., (1995) Partial molal adiabatic compressibilities of transfer of some amino acids and peptides from water to aqueous sodium chloride and aqueous glucose solutions Thermochim.Acta 175-183.

6. Banipal, T.S., Singh, G., (2004) partial molar and viscosities of some amino acids in aqueous electrolyte and non-electrolyte solutions J.Indian. Chem. Soc.81.126.

7. Hedwing, G.R., Hoiland, H., (1996) thermodynamic properties of peptides solutions. J.Solution.chem.25.1041- 1053.

8. Soto, A., Arce, A., Khoshkbarchi, (2003) Apparent molar volume isentropic compressibility, refractive index and viscosities of dl-alanine in aqueous Nacl solutions J. Solution Chem, 32.53-63.

9. Badarayani, R., Kumar, A., (2004) Effect of Tetra-N- Alkylammonium Bromides on the volumetric properties of Glycine-L-alanine and Glycylglycine at T=289.15K J. Chemical Thermodynamics, 36,49.