Drochioiu Detn AA Biostructure RoBioLett 2001

7/31/2019 Drochioiu Detn AA Biostructure RoBioLett 2001

1/11

Roum. Biotechnol. Lett. Vol. 6 ., No. 2 , 2001, pp. 155 - 165Copyright 2001 Bucharest University, Center for Research in Enzymology

and Biotehnology, Roumanian Society of Biological SciencesPrinted in Romania. All rights reserved

ORIGINAL PAPERS

The Breakdown of Plant Biostructure followed by Amino

Acids Determination

G. DROCHIOIU*, V. UNEL*, C. ONISCU**, CRISTINA BSU***,MANUELA MURARIU**

* Faculty of Chemistry, Al. I. Cuza University, 11 Carol Avenue, Ro-6600 Iassy,Romania, telefax 004032201201** Faculty of Industrial Chemistry, Gh. Asachi Technical University, 71 MangeronAvenue, Ro- 6600 Iassy, Romania*** SC Antibiotice SA, Iassy, 11 Valea Lupului Street, Ro- 6600 Iassy, Romania

Received: 2nd April, 2001; Accepted: 10th April, 2001

Abstract

A very simple, highly sensitive, fast, reproducible, and specific method for theinvestigation of the state of plant biostructure is proposed. It consists of three parts: (i) plantbiostructure alteration, or the screening of the plants being investigated, (ii) the extraction ofamino acids in a 1 M sucrose solution, and (iii) amino acids analysis using a ninhydrinreagent or by another procedure. In the simplest case, the absorbance of the colorednynhidrinamino acids solution is read at 516 nm. The color system obeys Beers law in therange of 4 - 40 g mL-1 mixture of amino acids. The extraction efficiency of the 1 M sucrosesolution and the molar absorptivity (1.758 x 103 L mol-1 cm-1) are evaluated. All otherimportant analytical parameters were studied and the method was applied to follow thebreakdown of some plant biostructure. The method was found suitable to demonstrate for thefirst time the role of hypoxia on the plant biostructure.

Keywords: biostructure breakdown; amino acids determination; hypoxia

Introduction

According to the biostructural theory, the matter within the living organisms consistsof two forms: biostructured matter (biostructure) and coexistent molecular matter [1].The distribution of different substances (water, ions, enzymes, etc.) between the

molecular matter and the biostructured matter has been studied so far through thesqueezing procedure [2,3]. This procedure consists in squeezing living biologicalmaterial under hydrostatic pressures and in collecting the sap resulting from it; this sap ismade up of free water and the substances dissolved in it. Conventionally, this sap is


2/11

G. Drochioiu, V. unel, C. Oniscu, Cristina Bsu, Manuela Murariu

called vacuolar sap and represents the free components of the coexistentmolecular matter in the

biological material being studied [4]. The remaining residue contains biostructuredmatter, which disintegrates after its death, releasing the water and hydrosolublesubstances contained. The squeezing procedure has been used to investigate the

distribution of water [5], amino acids [6,7], sugars, asparaginase [8], L-alanine --keto-glutarate aminotransferase, phosphomonoesterase [9], etc. in the biostructured matterand the coexistent molecular matter of cabbage leaves and small corn plants.

In the case of pollen or plant seeds, the squeezing method cannot be used because ofthe too low content in water. Therefore, another method, which was called extractionmethod has been devised [4]. It consists in treating a certain amount of biologicalmaterial with a certain amount of distilled water, slightly stirring for a certain timeinterval and separating the extract by centrifuging. The extract contains the componentsof the coexistent molecular matter [4].

The biostructured matter features a remarkable characteristic of breaking downpartially and reversibly. This phenomenon may be developed under the action ofmetabolic inhibitors [10], as a heat effect, by electrical stimulation [11], u.v. irradiation,or may occur spontaneously, and under various physiological, pathological andexperimental conditions [12]. Therefore, the state of living matter must be regarded as

being dependent on the state of biostructured matter [12].At present, there is no specific method to quantify the breaking down of the

biostructured matter in the living bodies. Neither the squeezing method nor theextraction one were used to serve this purpose. They are either sophisticated and difficultenough to be used or less fast making possible an additional breaking down of the plant

biostructure. Nevertheless, the use of these methods demonstrated that the breakdown ofthe biostructure results in the release of a variable amount of water, amino acids, sugars,etc [12]. Consequently, a simple method to follow the state of the living organisms,which is dependent on the integrity of their biostructure, was elaborated. Therefore, this

paper refers to a spectrophotometric assay of amino acids extracted by a 1 M sucrosesolution after their release from the plant biostructure being altered.

Various reaction conditions were studied, optimized and applied for the investigationof the state of biostructure in seeds, plantlets and leaves.

Materials and Methods

Apparatus.A Carl Zeiss Spekol spectrophotometer with 1 cm matched cells was usedfor all spectral measurements. Calibrated glassware, weighing bottles, and a water-bathwere used.

Reagents.All chemicals used were of analytical reagent grade and all solutions wereprepared with distilled water.

Treatment solutions.

Sodium azide, 0.005%, 0.05%, and 0.5%, respectively, NaN3 solution.Dinitro-o-cresol, 0.1% solution of 2,4-Dinitro-o-cresol (DNOC).Potassium cyanide, 1% solution.

Roum. Biotechnol. Lett., Vol. 6, No. 2, 155 - 165 (2001)


3/11

The breakdown of plant biostructure followed by amino acids determination

Extraction solution.. A 1 mol l-1 sucrose solution was prepared solving 342 g sucrosein 1 L of distilled water.

Ninhydrin reagent. 0.4 g ninhydrin and 0.4 g Cd(NO3)24H2O were dissolved in 25mL of buffer solution pH 5.5 in a 100 mL measuring flask. Then, glycerin was added tothe mark. The solution is effective only on the day it is prepared.

Buffer solution. 54.4 g sodium acetate was dissolved by heating into about 50 mLdistilled water and, after cooling, 10 mL glacial acetic acid and, then, distilled waterwere added to 100 mL. The pH value of this solution should be 5.5. If necessary, the pHcould be corrected with sodium hydroxide or acetic acid.

Alcohol solution, 80 % (v/v).Standard amino acid solutions. A 1 mg mL-1 stock standard solution of amino acid

was prepared by dissolving 0.100 g of each amino acid in 100 mL distilled water.Working standard solutions were prepared by appropriate dilution. Calibrated curveswere plotted with alanine.

Other amino acid solutions. In order to check the proposed method, some solutions of40 g mL1 mixture of different amino acids were analyzed.Biological materials.Seeds of maize (commercial variety, 1000 seeds weighed 330 g)

and wheat (Henika spring wheat variety, 1000 seeds 37.2 g), harvested in 1999,purchased from the Agricultural Station of Suceava, were used for all determinations.Some seeds germinated for 3 days and other ones were used to obtain 14 days old

plantlets and leaves.Background. The proposed method consists of three parts: (i) plant biostructure

alteration, or the screening of the biological material being investigated, (ii) theextraction of amino acids, and (iii) amino acids analysis.

Procedure. The biostructure of both untreated cereal seeds and treated ones withdifferent inhibitors or by heating at different temperatures was investigated by the

proposed procedure. Germinated seeds, as well as plantlets and leaves resulted weretreated similarly. Thus, samples of 4 g wheat seeds and 10 g maize seeds were treatedwith 10 mL or 25 mL, respectively, of treatment solution for 1 lour. A blank withdistilled water was carried out. Then, the seeds were taken out, cleaned out with filter

paper, and immersed in 1 M sucrose solution or water for 1 hour. The amino acids fromthe extraction solution were then treated with ninhydrin reagent.

Young maize and wheat plants (3-5 g) were harvested from the seed and immersedwith their basis in 10 mL of water (blank) or treatment solutions, generally, for 1 hour.

Also, during some experiments, samples of leaves were cut in 1 cm pieces andimmersed in the treatment solution for 1 hour.

Then, the treated samples were cleaned out with filter paper and introduced carefullyinto adequate test tubes. The extraction solutions (10 or 20 mL) were added into theslanting tubes. These tubes were rolled easily from time to time, during the extraction

procedure.Analysis. A 1 mL of extraction solution containing amino acids extracted from seeds

or plantlets was pipetted into a test-tube, into which a 1 mL ninhydrin reagent wasadded. The mixture was stirred vigorously. The test-tube was kept for 60 min in a

boiling water-bath at 100oC, then cooled to the room temperature and 5 mL of alcoholsolution added.

The tube was stirred again. The absorbance of the colored solution was read at 516


4/11


nm in 1 cm cuvettes. The same procedure was followed for a blank, which remainedcolorless or pink under these conditions.

The calibration scale was prepared over the range 4-40 g alanine mL-1.The squeezing method and the water extraction one [2,4] were used for comparison.Statistics. The standard deviation (S), standard deviation of the mean (sx), correlation

coefficient (r), and t and F parameters were calculated in order to compare the threemethods.

Results and Discussion

Calibration curves. The absorbance was proportional to the concentration of alanineover the whole measured concentration range from 4 to 40 g mL1, where linearcalibration graph was obtained (Figure 1). When the other amino acids were used,different calibration graphs were obtained; the highest absorbance values were observed

in the case of glycine, and the lowest for hydroxyproline (Table 1). Therefore, becauseof the

Table 1 Absorbance and the molar absorptivity of some amino acids in theninhydrin reaction conditionsa

Amino acid Absorbance Molar absorptivityAbsorbance (%,21 hours later)

Alanine

LysineTryptophanPhenylalanineArginineIsoleucine

NorleucineLeucineGlycine-Aminobutyric acid

-Aminobutyric acidSerineCystineHistidineMethionineCysteic acidProline4-HydroxyprolineAsparagine

Valine

0.790 0.035

0.447 0.0180.332 0.0160.415 0.0140.372 0.0180.480 0.0170.542 0.0200.551 0.0231.150 0.0450.628 0.030

0.449 0.0250.862 0.0350.430 0.0150.573 0.0230.257 0.0180.435 0.0160.235 0.0170.055 0.0140.505 0.022

0.501 0.023

1.758103 77.9

1.631103

67.31.696103 80.11.717103 55.81.619103 79.41.571103 56.81.775103 65.61.806103 75.72.156103 85.81.616103 78.8

1.156103

65.52.263103 89.82.580103 90.72.224103 88.70.957103 67.52.032103 73.20.758103 55.80.181103 44.51.662103 71.4

1.66010

3

72.3

7.3

5.35.010.03.02.06.74.3

12.0+2.8

7.45.0+3.72.1+6.02.0+2.8+3.7

10.7

2.0Mean 0.500 0.022 1.640103 72.1 3.3

a Mean of four replicate analyses; 40 g mL1 amino acid.



5/11


different composition in amino acids of the biological samples, standard curve might beplotted with the most adequate amino acid for the sample being analyzed. The molarabsorptivity of this amino acid and that of the mixture of the amino acids from extractionsolution must be as close as possible. Alternatively, the amino acid used for calibration

should be specified.A solution containing 40 g mL1 mixture of different amino acids similar to thatfrom wheat seedlings were analyzed by the proposed method (Table 2). Molarabsorptivity for mixture of amino acids from wheat was calculated to be 2.047103

(Mean formula weight 137.4; molar absorptivity values were taken from Table 1).Therefore, the most suitable amino acid to be used in the case of wheat amino acids ishistidine (Absorbance 0.573; molar absorptivity 2.224103 L mol-1cm-1).

Effect of various reaction conditions. The effect of reagents, reaction time andtemperature on the color development was studied after extraction of the amino acids in

1 mol L

-1

sucrose solution. A minimum of 1 mL ninhydrin reagent was found to besufficient for complete reaction of amino acids. The resulting compound has anabsorption maximum at 516 nm (500-530 nm).

A minimum of 60 min were needed for full color development at 100 oC. Theabsorbance of the colored solution increased 7.5-fold when time of the reaction wasincreased from 5 min to 60 min (Figure 2). It decreased again just 2-fold when test tubeswere kept for 210 min in a boiling water-bath, indicating a decomposition of the dyeformed.

Figure 1 Standard curve for the determination of

amino acids in the extraction sucrose solution

y = 0.0198x - 0.0025

R2

= 0.9994

0

0.2

0.4

0.6

0.8

0 10 20 30 40

Concentration of alanine, g ml-1

A

bsorbance


6/11


Also, the effect of ninhydrin concentration was investigated up to 1 % in theninhydrin reagent. A 0.4 % ninhydrin concentration was needed for maximumabsorbance. Also 0.4 to 1.0 % of ninhydrin caused no change in the absorbance, exceptthe fact that the usage of more concentrated reagents resulted in a greater stability intime of the colored solution.

The absorbance of the colored solution obtained from alanine increased almost 12-fold when temperature of the reaction was increased from 55 oC to 100 0C (Table 3). Theother amino acids showed different values for the absorbance in the same conditions.Glycine reacted easier while isoleucine heavier when temperature of the reaction wasmaintained at the lowest values (Table 3). This suggests that strongly activated carbonylgroup of ninhydrin attacks electrophilically the amino group of amino acids and that thereaction rate increases due to the electronic effect (IS) of the radicals from amino acidand decreases because of the steric (hindering) factors. The last aspect wasdemonstrated by the molar absorptivity of isoleucine, norleucine and leucine (Table 1).

Table 2 The absorbance of a mixture of amino acids similar to that from the wheat sap

Amino acid Concentration(g mL-1)

Relative absorbance

CysteineAsparagineHistidine

Aspartic acidGlycine

SerineGlutamic acid

ThreonineAlanineTyrosine

MethioninePhenilalanine

Leucine

1.2515.972.711.251.40

0.430.400.955.961.471.405.411.40

0.0270.2010.0390.0160.043

0.0100.0050.0130.1180.0150.0330.0560.020

Mean 40.00 0.596

Interferences. Effect of co-existing species was examined with 40 g mL-1 ofalanine solution. The method was found to be free from most of the interference.

Nevertheless, ammonia, amines etc. named ninhydrin-positive compounds react withninhydrin as well. In the conditions of plant biostructure investigation by the proposed

procedure, all of the ninhydrin-positive compounds were found not to interfere seriouslybecause of their low concentrations. Nevertheless, to eliminate any interference and toincrease in specificity of the assay, a blank with the untreated sample should be carriedout.

Reproducibility and sensitivity. The proposed method is reproducible and the colorsystem was found to obey Beers law in the range 4-40 g mL-1 of alanine in the 1 Msolution of sucrose. The molar absorptivity and Sandells sensitivity [13] were found to

be 1.758 x 103 (77.9) l mol1cm1 and 0.015 g cm-2 respectively.Roum. Biotechnol. Lett., Vol. 6, No. 2, 155 - 165 (2001)


7/11


In order to characterize the precision of the proposed method, a sample of alaninecontaining 20 g mL1 alanine was analyzed by the two methods. The result wasexpressed as the mean of six replicate analyses. The F parameter (F = S1/S2 =0.20874/0.16525 = 1.26)showed that the two methods are similar (Table 4).

Table 3 Dependence of the color intensity of the solution of the aminoacid ninhydrin dye on the temperature of reaction.

Amino acidAbsorbance

55oC 65oC 80oC 90oC 100oC% % % % % 100%

AlanineGlycineLeucineIsoleucine

NorleucineLysine

8.5018.207.452.97

5.675.94

22.4036.0720.3710.10

18.0015.98

47.0546.1536.8029.97

34.8931.82

71.1073.2468.8968.54

71.8663.36

100100100100

100100

0.7901.1500.5510.480

0.5420.447

Extraction efficiency. The release of amino acids from the leaves of wheat seedlingswas researched after the treatment with sodium azide in various concentrations. Avolume of 35 mL sap was released by the squeezing method up to 0.005% NaN3 and 58mL sap from 100 g wheat leaves when a 0.5% sodium azide solution was used (Table 5)

Figure 2 The dependence of the color intensity of

ninhydrin-amino acid dye on the reaction time interval

0

0.2

0.4

0.6

0.8

1

0 50 100 150 200 250

Time, min

Absorbance

100 oC, 40 g ml-1


8/11


Also, this sap contained 73.1 to 543.6 mg mixture of amino acids. Water extractionmethod [4] gave the smallest values for the amino acids released and the proposedmethod higher ones.

The correlation coefficients between the proposed method and the known ones werehigh enough (r = 0.997 and 0.991 respectively). All of the three methods showed the

same thing: the amino acids may be found both in biostructure and in vacuolar juice,their release is progressive depending on the increase in the concentration of inhibitor.Simultaneously, the content in free amino acids of both untreated leaves and the treatedones with 0.5% sodium azide solution was determined colorimetrically [14,15]. Similarvalues for the content in free amino acids (1.056% and 1.010% respectively) wereobtained.

Table 4 Evaluation of the precision of the proposed method compared withthe water extraction method [4] for the determination of alanine

Parameter Proposed methodg mL1

Water extraction methodg mL1

CSsx

19.93 0.240.20874

0.093

19.95 0.190.16525

0.073

In order to prove the extraction efficiency of amino acids from maize seedlings,samples of 5 g plantlets were extracted (four repetitive determinations) with 50 mLof

1 M sucrose solution for 30 min. Extraction was repeated from 30 min to 30 min formaximum 18 hours. Separately, some samples were extracted similarly, still the samefour samples were extracted for 30 min, cleaned up, extracted again for 30 min, etc.(Table 6). The maximum amount of released amino acids was found for the first 30 minof the extraction. It decreased slowly for the next 150 min probably because of the

penetration of the sucrose solution into the plant cells. The last extraction demonstrated

Table 5 Extraction efficiency of amino acids from wheat seedlings (100 g leaves;extraction time, 24 h)

Parameter Sodium azideconcentration

Thesqueezing

method

Water extractionmethod

The proposedmethod

Sap (mL)

Amino acids

(mg)

0%0.005%

0.05%0.5%

0%

0.005%0.05%0.5%

35354858

71.3

85.1249.6543.6

19.3

23.341.5110.2

34.7

41.7115.4260.1


9/11

Roum. Biotechnol. Lett., Vol. 6, No. 2, 155 - 165 (2001)The breakdown of plant biostructure followed by amino acids determination

that an additional breaking down of the biostructure from maize seedlings had occurred.

Application of the method

Wheat seed samples. Germinated seeds of wheat (100 seeds) were treated with 10 mLwater or 10 mL of 1 M sucrose solution for 1 hour. The sucrose solutionextracted 48.30 3.4 mg % amino acids (expressed as alanine), while water just 26.66 2.1 mg %. The experiment was repeated with wheat seedlings treated with water orDNOC, cleaned up with filter paper, and then extracted for an hour with water or sucrosesolution (Table 7). Sucrose solution extracted a bigger amount of amino acids comparedwith water, making possible to differentiate the effect of this inhibitor on the biostructure

breakdown.

Table 6 The effect of extraction time on the amount of amino acids releasedfrom maize plantlets (expressed as mg alanine from 100 g plantlets)

Extraction time (min) Repetitive extractions Single extraction

306090

120150

1080

68.0 1.418.0 0.75.4 0.82.6 0.72.0 0.3

36.6 0.6

68.0 2.165.4 1.764.3 1.963.2 3.161.8 1.2

115.4 3.5Total 132.6 115.4

Effect of cyanide.Samples of 3 g maize seedlings were kept in 10 mL of 1% KCNsolution for an hour against 10 mL water. Then, the samples were taken out, cleaned upwith filter paper and kept for 30 min in a 20 mL of 1 M sucrose solution. The releasedamino acids (expressed as mg % alanine) increased from 58.00 1.45 to 226.40 13.41mg alanine % when KCN was used. No other alterations of plants were observed exceptthe mentioned increase in the amino acid concentration from the extraction solution.

Table 7 The effect of DNOC on the breaking down of wheat biostructure and theopportunity to follow it by two specific methods

Treatment Extraction solution Released amino acids (mg %)a

H2ODNOCH2O

DNOC

H2OH2O1 M sucrose

1 M sucrose

31.86 10.7261.62 17.2038.80 10.82

157.98 12.14a mean of six repetitive analyses



10/11


Advantages of the method. Among the outstanding features of the method is the largenumber of determinations, 20 single samples determinations, per 8 h working day.

Only common, inexpensive equipment is necessary; no special training is neededexcept skill in handling simple, chemical glassware and reagents and in reading a

colorimeter. The procedure requires just a few reagents easily available, namely aninhydrin reagent and a sucrose solution, unlike the standard procedure that makes useof a laboratory press. This method may quantify the alteration of plant biostructure dueto hypoxia, effect of some inhibitors and noxae, or by other means.

Conclusion

The proposed method is useful to follow the breaking down of plant biostructureusing a very simple determination of amino acids in 1 M extraction solution of sucrose.

The method can also be used for environmental analysis, and for biochemical work, too.Only common, inexpensive equipment is necessary; no special training is needed exceptskill in handling simple, chemical glassware and reagents and in reading a colorimeter.

References

1. E. MACOVSCHI,Biostructure, Ed. Academiei, Bucharest, (1969) p. 190.2. E. MACOVSCHI, V. FRUNZETI,Rev. Roum. Biochim., 5, 3-6, (1968).3. E. MACOVSCHI, V. ELEFTERESCU, I. CORNOIU, C. MUOLAN, O. GOZIA,

St. cerc. Biochim,.15, 377-389 (1972).4. C. IORDACHE,Rev.Roum. Biochim., 22, 19-21 (1985).5. R. STNESCU, St.cerc. Biochim.,15, 101-105 (1972).6. I. CORNOIU, St.cerc. Biochim.,15, 21-24 (1972).7. A. MIHIESCU, St.cerc. Biochim.,15, 111-115 (1972).8. I. CORNOIU, St.cerc. Biochim.,15, 177-181 (1972).9. M. RABEGA, St.cerc. Biochim.,15, 201-205 (1972).10. E. MACOVSCHI, St.cerc. Biochim.,11, 213-217 (1968).11. E. MACOVSCHI,Rev.Roum. Biochim., 17, 183-191 (1980).12. E. MACOVSCHI,Rev.Roum. Biochim., 21, 3-11 (1984).13. D.M. HOLLAND, F.F. MCELROY, Environ. Sci. Technol. 20, 1157-1161

(1986).14. G. DROCHIOIU, M. CRISTEA, D. LAZAR, D. MURARIU, Cercet. Agron. in

Moldova, Iassy, 3, 16-20 (1990).15. G. DROCHIOIU,Ph.D. Thesis, University Al.I.Cuza Iassy, 35-38 (1997).


11/11


Documents

Drochioiu Detn AA Biostructure RoBioLett 2001