Optical methods of estimating proline concentration in natural biologic environments icon

Optical methods of estimating proline concentration in natural biologic environments




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Optical methods of estimating proline concentration

in natural biologic environments

S.G. Guminetskiy, A.V. Motrich, S.S. Rudenko, S.B. Gritsuk


Chernivtsy National University named after Yu. Fedjkovych

motrich@gmail.com


ABSTRACT( COR09-COR01-49)

A method for determining optical proline concentrations in samples of natural waters, based on the measurement value angle of the plane of polarization of radiation of a certain wavelength λ, which has been studied by the liquid thickness l = 5 sm was developed.

Three wavelength λ = 400, 450, 500 nm were used. A series of reference samples was produced: aqueous solutions of L and D forms proline of a known concentration within c = (0.001 – 0,1) %.

Based on these studies, for each λ gauge dependent quantities of angle α = f (c) was built. Using them and conducting measurements of angles α for the real natural environments, the possibility of determination of the concentration of proline in them was actualized.

It was determined that in waters within the city of Chernivtsy and its surroundings occured proline of two forms: L and D, the concentration of which is within c = (0,005 - 0,094)%


^ Key words: optical activity, proline, natural reservoirs



  1. INTRODUCTION


According to literary sources, it is known that amino acid proline (pirrolidyn-2-carboxylic acid) is a universal stress-protective and antioxidant compounds and it plays an important role in the survival of higher plants and algae under stress. Because of stressful factors of free proline, the content in these organisms increases in the tens and sometimes hundreds of times.

When in the contaminated river ecosystems, especially in the warm days, oxygen in the upper layer of water appears almost exhausted, and because of that there is a massive loss of organisms that live in surface waters (so-called "summer refresh"), then the previously accumulated during the growing stress proline with damaged cells gets into the water and its concentration and chiral properties may serve as markers of pollution level of river ecosystems and degradation of biota [1, 2].

It is therefore important to have a method for determining the concentration of proline in natural waters, in this case the most commonly used are biochemical methods [3].

Physical, including optical methods, according to literary sources are not used. In this regard, the task of this work is the development and testing of the optical method of determining the concentration of proline in samples of natural waters.



  1. ^ THE OBJECTS AND METHODS OF INVESTIGATION


As objects of research at first served model water solutions proline amino acids L and D forms (Fig. 1) of a given concentration, which were in the range c = (0.01 - 1) • 10-1%, which corresponded according to literary data [1, 2, 4, 5] to the possible content of the amino acids in the contaminated water.

Then water samples were investigated in small rivers of Chernivtsy. The value of the angle of the plane of polarization for three plane mirror radiation wavelength λ = 400, 450, 500 nm was measured.

Tests were conducted on laboratory polyarymetrychniy plant, which provided a minimum focusing plane of polarization angle ± α = 3 `.




L-forms D-forms


Fig. 1 Spatial isometric L and D forms of extraction proline.

Functional optical scheme given in Fig.2.




Fig. 2 Scheme of polarization laboratory settings


Here: 1 - light source (halogen lamp), 2 - condenser; 3-5 - MUM monochromator; 6,8 - Diaphragm 9 - a ditch with the study solution, 7 - polarizer, 10 - meter, 11 - circle, 12 - receiver radiation (FEP) 13 - digital voltmeter, 14 - stabilized power supply, 15 - high-voltage power supply for FEP; D1, D3 - turning mirrors; D2 - dispersive element (spherical diffraction grating).


Required wavelength was allocated by fast MUM monochromator; study sample was put between the polarizer and analyzer (Glan-prism); as a radiation detector served a photo multiplier, the value of the electric signal of which was registered with the help of a digital voltmeter.

Measurement value angle of the plane of polarization of the samples was conducted by the zero point: first, in the course of radiation a ditch thickness l = 5 sm with distilled water is placed and by crossing the plane of polarization of the analyzer and polarizer was fixed minimum counting on the digital voltmeter, and on a scale limbu, which secured analyzer, a value corresponding angle α0; then in progress rays was put same ditch with the study solution and by rotation of the analyzer was again the minimum count on the digital voltmeter and the corresponding value of angle on a scale αp limbu. Size angle find the plane of polarization as ± α = αp - α0. In order to improve the accuracy of determining its value was in α as the average of ten measurements for the concentration and fixed λ.


^ 3. STUDY OF OPTICAL ACTIVITY OF MODEL SOLUTIONS PROLINE


We investigated five solutions of proline L and D forms of different concentrations. The research results are given at Table 1.


Table 1. Value angle α (°) plane of polarization of solutions proline.

С , %

L- proline

λ = 400 nm

λ = 450 nm

λ = 500 nm

0,1

-0,7

-0,64

-0,64

0,05

-0,56

-0,42

-0,34

0,01

-0,38

-0,24

-0,24

0,005

-0,24

-0,12

-0,12

0,001

-0,16

-0,1

-0,04

С , %

D- proline

λ = 400 nm

λ = 450 nm

λ = 500 nm













0,1

1,36

0,47

0,26

0,05

1,34

0,44

0,18

0,01

0,9

0,41

0,14

0,005

0,77

0,34

0,1

0,001

0,51

0,28

0,08



We see that with decreasing of λ and with increasing of the concentration in solution of proline the angle of the plane of polarization increases. This corresponds to a theoretical understanding of the optical activity of liquids [6, 7]. Based on the results for each λ a gauge dependence α = f (c) (Fig. 3) was built, here the axis is divided into logarithmic scale.


Fig. 3 Comparison of the dependence α = f (c) for L, D-proline form (on a scale abscissa logarithmic scale)


A view of dependency forms of L-proline and proline D-form, represented in Fig. 4 and 5.





Fig. 4 The dependence α = f (c) to form L-proline (on a scale abscissa logarithmic scale)





Fig. 5 The dependence α = f (c) to form D-proline (on a scale abscissa logarithmic scale)


Further, they can be used to determine concentrations of proline in natural aquatic environments. Method of determining the values of the concentration are represented at Fig. 6 and Fig. 7, for wavelength 450 nm, where there is the slightest fluctuations in the measurement of multiple proline solution in distilled water with different concentrations, that is within s = (0.001 - 0.1)% for D and L forms of proline.




Fig.6 methodology for determining the values of the concentration of L-Proline for λ = 500





Fig. 7 Methods of determining the values of the concentration of D-Proline for λ = 500.


However, in these environments, except proline may be other amino acids. But it is known [8], their solubility in water is much less than the proline for H20 solution at t = 250С (or distilled water at the same temperature), proline has a solubility of 162.3 (mg / 100 ml), and soon has a value of solubility amino acid treonyn 20.5 (mg / 100 ml).

In addition, proline has the largest optical activity: for example, to form L-proline in the water value of the specific rotation for sodium D line is 86,2 ° for solution in H20 at t = 250С (or distilled water at the same temperature), and near the optical activity, namely the value of specific rotation for sodium D line has the amino acid treonyn, 28.50. Therefore, when studying the optical activity of natural water reservoirs possible presence of other amino acids can be neglected.


^ 4. Investigation of the optical activity of samples of real natural water reservoirs


Research was carried out by analogy with the solution of amino acids. Water samples were taken from small rivers within the city of Chernivtsy, as well as in the suburban area near industrial enterprises.


^ Table 2. Value angle α (°) and proline concentration c (mg%) in natural waters



Place of sampling

α, °

с, mg%

Molnytsa River

1.

Auto-refueling complex

-0,88

152,8

2.

Brickworks №3

-0,08

2,.2

3.

Garages

0,06

0,1

4.

Emergency spill of the “Chernivtsivodokanal”

0,22

3,7

5.

Chernivtsy Woodworking Complex

-0,10

2,5

Koroviya River

6.

Auto-refueling complex

-0,44

53,3

7.

Prydnistryanska Station of Horticulture

-0,14

5,1

Klokuchka River

8.

Section of the destroyed canalization collector of the “Chernivtsivodokanal” Enterprise

0,18

10,4

Shubranets River

9.

Chernivtsi Oil and Fat Complex

0,18

10,4

10.

Kalynivka Market

0,18

10,4



Let’s determine the concentration of L or D proline in the studied environment by conducting measurement of angles α and using calibration graphs for the corresponding λ which are given on Fig. 3 - 7 (depending on the sign of α).

For example, the results of such investigation with the use of these calibration graphs for λ = 500 nm are given at table 2. Similar results can be obtained using calibration graphs for λ = 400 nm and λ = 450 nm.

The resulting concentration values are obtained by averaging of three values received for each λ values c.

From table 2 follows that samples of water of small rivers of Chernivtsy and its neighborhoods show a left and right turning activity. While for plant and animal organisms are more typical is a left form rotational proline, and for bacterial - D-proline, it can be confirmed that in areas of small rivers, where the D-Proline dominates, a bacterial contamination predominates, and where the dominant is L-proline we see a contamination of plant and animal origin.

It means that a mark of the angle of the plane of polarization indicates the optical activity of water samples and it can be used to identify the nature of pollution of natural water.

In addition, Table 2 shows that the degree of water pollution in rivers near the different companies is different: it is the largest near the petrol sector.

Table 3 compares the results of the concentrations of proline by the proposed optical method (averaging three λ) and biochemical methods are performed by the scheme given in [3].


^ Table 3. Value proline concentration c (mg%) in natural water obtained by different methods



Place of sampling

Optical method

с, mg%

Biochemical method

с, mg%

Molnytsa River

1.

Auto-refueling complex

149

141

2.

Brickworks №3

1,8

1.0

3.

Garages

0,1

0.08

4.

Emergency spill of the “Chernivtsivodokanal”

3,5

3.9

5.

Chernivtsy Woodworking Complex

2.5

2.0

Koroviya River

6.

Auto-refueling complex

50,9

56.9

7.

Prydnistryanska Station of Horticulture

49

44

Klokuchka River

8.

Section of the destroyed canalization collector of the “Chernivtsivodokanal” Enterprise

8

5

Shubranets River

9.

Chernivtsi Oil and Fat Complex

8,9

9.4

10.

Kalynivka Market

11,1

12.5


We see that the values of concentrations agree with each other and better than they are bigger.

In fact, using an optical method for determining the concentration of proline in samples of natural water is enough to measure the value of the angle of the plane of polarization of only one of the above three wavelengths.

Optimal in this case, we see λ = 450 nm. The fact that for λ = 500 nm, as shown by the study, the value of α is the lowest compared with other λ, so the accuracy of their determination is the worst.

On the other hand, due to a significant decrease transmittance of radiation and the receiver sensitivity for λ = 400 nm accuracy of fixing the minimum signal to a digital voltmeter to zero, the method is worse than the other λ, which in turn again lead to a deterioration of accuracy in determining α


5. CONCLUSION


From the results obtained in the work can make the following conclusions:

1. The proposed optical method can be successfully used to assess the degree of contamination of natural water bodies as a simpler and less laborious than using for this purpose the biochemical methods.

2. With the help of optical method we can evaluate not only the degree of contamination of natural water reservoirs, but also we can identify the nature of the contamination.


6. REFERENCES


  1. Yakubke H.-D. Amino acids Peptides Proteins. / H.-D. Yakubke, JE. Eshkayt. / / Moscow. Peace. - 1985. - 84 pp.

  2. Volkov R.V. Ellipsometric study of optical properties of the cornea: summary dissertation on competition of a scientific degree of candidate of physical and mathematical sciences: Specialty 01.04.03 "Radiophysics" / R.V. Volkov. / / Volgograd. - 2006. – 20 pp.

  3. Bates L.S. Rapid determination of free praline for water-stress studies. / L.S.Bates, R.P. Waldren, I.D. Teare. // Plant and Soil, – 1973. – Vol. 39. – P. 205 – 207.

  4. Higashi Y. N.- M. The presence of high concentrations of free d-amino acids in human saliva. / Yoko Nagata Masatoshi Higashi, Yutaka Ishii, Hiroaki Sano, Minoru Tanigawa, Kumiko Nagata, Kazuma Noguchi and Masahiro Urade. // Life Sciences. – 2006. – Vol. 78. – P. 1677-1681.

  5. William R. Schwan. Low-proline environments impair growth, proline transport and in vivo survival of Staphylococcus aureus strain-specific put mutants. / William R. Schwan, Keith J. Wetzel, Timothy S. Gomez, Melissa A. Stiles, Brian D. Beitlich and Sandra Grunwald. // Society for General Microbiology. Microbiology, – 2004. – P. 1055-1061

  6. Vellyuz L. Optical circular dichroism. / Vellyuz L., M. Legrand, M. Hrozha / / Moscow. Peace. - 1968. - 315 pp.

  7. Born M. Principles of optics. / Born M., Wolf JE. / / Moscow. Peace. - 1970. - 855 pp.

  8. Master A. Biochemistry amino acids / A. Master / / Moscow. Science. - 1985. - 315 pp.

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