- Email: [email protected]
Molecular spectroscopic analyses of gelatin
Spectrochimica Acta Part A 81 (2011) 724–729
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Molecular spectroscopic analyses of gelatin Medhat Ibrahim ∗ , Abdel Aziz Mahmoud, Osama Osman, Mohamed Abd El-Aal, May Eid Spectroscopy Department, National Research Center, 12311, Dokki, Cairo, Egypt
a r t i c l e
i n f o
Article history: Received 24 April 2011 Received in revised form 30 June 2011 Accepted 3 July 2011 Keywords: Natural protein Amino acids FTIR DFT
a b s t r a c t The molecular structure of gelatin was studied using Fourier transform infrared spectroscopy FTIR. The spectrum is subjected to deconvolution in order to elucidate the constituents of the molecular structure. B3LYP/6-31g** was used to study 13 amino acids then the scaled spectrum was compared to those of protein in order to describe the contribution of each amino acid into protein structure. A special interest was paid to the NH and C O region. The reactivity of each amino acid was studied in terms of some important physical parameters like total dipole moment and HOMO/LUMO which describe the interaction of amino acid with their surrounding molecules. Results indicated that B3LYP/6-31g** model is a suitable and precise method for studying molecular structure of protein. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Amino acids are the building units of protein. Among the twenty naturally occurring protein is glycine. Glycine is the simplest amino acid, with no side chain. Therefore, glycine is of particular importance as a structural unit in some proteins like collagens, and in many metabolic pathways acting as a source of one-carbon units. In mammals, collagen contains over 30% glycine, and collagens make up 20–25% of the body’s protein [1]. Glycine is also essential as a metabolic precursor. Readily converted to serine and is involved in the synthesis of both creatine and glutathione. Additionally, glycine is an integral precursor of uric acid [2]. Moreover, Glycine is the most potent inhibitory neurotransmitter in the spinal cord [3]. Amphipathic residues such as tryptophan (Trp) are believed to play a critical role in the structure and function of integral membrane proteins. It is strongly believed that the common occurrence of Trp residues at the aqueous–lipid interface region of transmembrane channels is indicative of its importance for insertion and stabilization of the membrane channel [4]. Alanine is quantitatively the primary amino acid released by muscle and extracted by the splanchnic bed in post absorptive as well as prolonged fasted man. The hepatic capacity for conversion of alanine to glucose exceeds that of all other amino acids. Insulin inhibits gluconeogenesis by reducing hepatic alanine uptake. In contrast, in diabetes, an increase in hepatic alanine extraction is observed in the face of diminished circulating substrate. In prolonged fasting, diminished alanine release is the mechanism whereby gluconeogenesis is reduced. In circumstances in which alanine is deficient,
∗ Corresponding author. Tel.: +20 122727636; fax: +20 233370931. E-mail addresses: [email protected], [email protected] (M. Ibrahim). 1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2011.07.012
such as pregnancy and ketotic hypoglycemia of infancy, fasting hypoglycemia is accentuated [5]. The non-polar amino acid phenylalanine which is naturally present in breast milk of mammals, has attracted attention since chronic excess of phenylalanine in the blood circulatory system has been shown to be associated with deficiencies in the total myelin content both in the human phenyl ketonuric patient and in experimentally induced hyperphenyl alanemia. Assuming that alterations of macromolecular synthesis may be related to the mental retardation often associated with untreated phenyl ketonuria, several investigators have studied the effects of high levels of phenylalanine on neural protein metabolism [6]. Studies indicated that Phe reduced neurite outgrowth of the cortical neurons and inhibited neuronal survival. Furthermore, previous work has shown that Phe affected BDNF mRNA and protein expression. In addition, Phe inhibited the activity of ERK and Akt [7]. Cytosine is one of the main four bases found in DNA and RNA, derived from pyrimidine, it was found that DNA daily attack by oxygen reactive specious could cause deamination of DNA bases, resulting in a direct damage of DNA. It has been reported that there is a correlation between DNA duplex melting and cytosine deamination, indicating that deamination in double-stranded DNA is inversely related to duplex stability [8,9]. Fourier transform infrared spectroscopy is a powerful technique for elucidating molecular structure. It could be utilized in studying nano-materials [10]; polymers [11]; environmental samples [12–14]; biological interaction [15,16]. Both FTIR and molecular modeling were used to study glycine among other structures [17]. The aim was to follow up the vibrational characteristics of COOH. Later on amide as well as COOH and many functional groups were a topic of modeling work to assess their reactivity [18]. The possible interaction of alkali metals as well as heavy metals with protein was conducted using density functional theory method DFT [19]. Alanine was choosing as a model molecule for this interaction.
M. Ibrahim et al. / Spectrochimica Acta Part A 81 (2011) 724–729
The present work is conducted to study gelatin by FTIR. Some amino acids were studied by B3LYP/6-31g** model. The deconvolution of FTIR was conducted and compared with molecular modeling results to assign the vibrational spectrum of gelatin.
725
2.2. Instrumentations
2. Materials and methods
Jasco FTIR 430 Fourier transform infrared spectrometer was used for recording the obtained IR spectra. Spectra were recorded in a spectral range of 4000–400 cm−1 , resolution of 4 cm−1 and scan speed is 2 mm/s.
2.1. Reagents
2.3. Calculation details
Gelatin from bovine skin, Type B was obtained from Sigma– Aldrich (Steinheim, Germany).
Model molecules represent the following amino acids alanine (Ala), cytosine (Cyc), glycine (Gla), tryptophan (Try), phenyl ala-
Fig. 1. Optimized structures of the studied amino acids.
726
M. Ibrahim et al. / Spectrochimica Acta Part A 81 (2011) 724–729
Fig. 1. (Continued )
nine (Ph.Ala), asparagine (Asparg), aspartic (Asp), glutamine (Glu), histidine (His), leucine (Leuc), methionine (Meth), proline (Prol) and valine (Val) were constructed. All calculations were performed on personal computer using the Gaussian 03 program system [20] Geometries were optimized using density functional theory DFT, hybrid Becke 3-Lee–Yang–Parr (B3LYP) exchange correlation functional [20–23] with 6-31g** basis set. HOMO-LUMO frontier energies and vibrational spectra were calculated at the same level of theory. 3. Results and discussion For each amino acid the geometry is optimized at B3LYP/631g**, then vibrational frequencies were calculated at the same level of theory. The optimized amino acids are indicated in Fig. 1.
3.1. Calculated bond lengths and bond angles Table 1 shows the optimized parameters of the calculated amino acids. It is clear that among the studied amino acids, thirteen of them own only one NH2 group (amide) while asparagines and glutamine have two NH2 groups. Moreover, all the same thirteen amino acids have at least one carboxylic group, except cytosine. Geometrical parameters were represented for both amid groups (NH2 and COOH). The bond length of C O ranged from 1.20 to 1.23 A˚ which is nearly constant for all the studied amino acids. Some amino acids show identical NH bonds such as alanine, cytosine, glycine, tryptophan, phenyl alanine, histidine, methionine and valine, while, ˚ which is not Others show by difference in their NH about 0.01 A, a significant difference. So, the two bonds which consist the amide
M. Ibrahim et al. / Spectrochimica Acta Part A 81 (2011) 724–729
727
Fig. 1. (Continued ).
Table 1 B3LYP/6-31g** calculated bond lengths (Å) bond angles (◦ ) for the studied amino acids. LC Alanine Cytosine Glycine Tryptophan Phenyl alanine Asparagine Aspartic Glutamine Histidine Leucine Methionine Proline Valine
LC
O
1.21 1.22 1.20 1.21 1.21 1.23 1.21 1.22 1.21 1.21 1.21 1.21 1.21
O
1.21 1.21 1.21
NH1
NH2
1.02 1.01 1.01 1.02 1.02 1.01 1.01 1.02 1.02 1.02 1.02
1.02 1.01 1.01 1.02 1.02 1.02 1.02 1.01 1.02 1.01 1.02
1.02
1.02
NH1
OCO
OCO
122.4
1.01 1.01
groups are identical. Regarding the bond angles OCO angle ranged from 120◦ to 124◦ . While HNH angle ranged from 106◦ to 118.8◦ . Although the bonds distances of NH2 groups are identical, their angles are not. 3.2. The total dipole moments and energy gaps The calculated dipole moment can be expressed in terms of vector in three directions X, Y and Z. It is considered as being the measure of asymmetry in the molecule charge distribution. The dipole moment is derived from the first derivative which reflects the static behavior of the molecule. Table 2 indicates the calculated total dipole moment. The results show that the amino acids can be arranged in the following increasing order: Ph.Ala > Prol > Val > Ala > Meth > His > Asparg > Leuc > Glu > Try. Table 2 B3LYP/6-31g** calculated HOMO/LUMO energy (E) as eV, total dipole moment (TDM) as Debye.
Alanine Cytosine Glycine Tryptophan Phenyl alanine Asparagine Aspartic Glutamine Histidine Leucine Methionine Proline Valine
NH2
E
TDM
MPG
6.75 5.33 7.39 5.182 4.25 7.11 6.54 7.06 6 7.22 6.23 6.08 6.67
2.34 6.29 5.58 6.45 1.93 4.73 4.38 6.55 4.65 5.41 2.6 1.85 2.14
C1 C1 C1 C1
1.01 1.01
123.9 123.6 122.2 123.6 124.0 123.5 122.7 123.1 122.8 122.6 122.0
122.4
HNH
HNH
107.1 118.1 107.4 107.0 106.0 106.4 106.4 118.7 108.6 107.1 108.0
118.8 107.4
107.2
The difference between highest occupied molecular orbital HOMO and lowest unoccupied molecular orbital LUMO is calculated. These differences together with total dipole moment reflect the reactivity of a given structure with their surrounding molecule. The same arrangement could be made for the studied amino acids. The calculated band gap energy for the studied amino acids is arranged in the following order: Ph.Ala > Prol > Val > Ala > Meth > Hist > Asparg > Leuc > Glu > Gly.
3.3. Vibrational spectrum of gelatin The FTIR absorption spectrum and band assignment of gelatin is indicated in Fig. 2. Gelatin characteristic bands were described by Ibrahim et al. and Yin et al. [16,24] As seen in Fig. 2, the absorption bands of amide groups were located at 3434 cm−1 (NH stretching), 1638 cm−1 (amide I, C O and CN stretching), 1545.51 cm−1 (amide II) and 1243 cm−1 (amide III). These bands could be assigned to the characteristic bands of gelatin. The band at 2923 cm−1 represents the CH2 asymmetric stretching vibration which is followed by another one at 2853 cm−1 , which corresponds to symmetric stretching vibration. The CH2 bending and wagging vibrations raise two bands at 1452 cm−1 and 1337 cm−1 respectively. Finally the CH3 amide group appears at 1031 cm−1 while the COC band appears at 537 cm−1 . In our calculations we concentrate on both C O and N–H vibrations. The spectra indicate that although the geometrical parameters are identical, the frequencies appear different from each other. C O is ranged from 1738.0 cm−1 to 1884.0 cm−1 , while N–H ranged from 3487.2 cm−1 to 3607.3 m−1 . Theoretically, each band appears as a harp line while experimentally, the band appears broad. In order to demonstrate the contribution of amino acids in
728
M. Ibrahim et al. / Spectrochimica Acta Part A 81 (2011) 724–729 Table 3 B3LYP/6-31g** calculated C O and N–H symmetric frequency.
0,4
0,3
Absorbance
NH CH2
0,2
Amide I Amide II
0,1
CH3 COC Amide III
0,0 4000
3500
3000
2500
2000
1500
1000
Fig. 2. FTIR absorption spectrum of gelatin.
the protein structure, the deconvolution of the experimental spectrum was carried out. Extra assignment could be carried out with the deconvolution of the gelatin spectrum as in Fig. 3. Furthermore a correlation between calculated as well as FTIR spectrum could be found. Regarding the NH stretching in region 4000–3000 cm−1 . There are some NH bands which could be attributed to the presence of tryptophan, cytosine and phenyl alanine respectively. This could be further emphasized with the finger print of aromatic ring around 1000 cm−1 and the CH stretching for the aromatic ring at 3088 cm−1 . Regarding the theoretical results performed using B3LYP/6-31g** level of theory, the calculation of E for the thirteen amino acids (Table 2) show that cytosine is 5.3 eV, tryptophan is 5.2 eV, whereas, phenyl alanine is 4.2 eV. Therefore, these three aromatic compounds show the lowest binding energy values among the 13 studied amino acids. Thus, these three amino acids could be the weak points at which the breaking down of big alpha helix structure (collagen in our case) into the beta sheet (gelatin in our study) could occur (see Table 3). From the above results we can conclude that the two peaks of the NH stretching regions represent one aliphatic and one aromatic amino acid. The error in this method is systematic and could be overcome by scale factor to match both experimental and theoretical data. Results may prove that the two main amino acids in gelatin molecule are tryptophan and glycine. The tryptophan is an aromatic amino acid and the glycine is an aliphatic amino acid.
Absorbance
1,0
0,5
3500
3000
2500
2000
1500
1000
-1
Wavenumber [Cm ] Fig. 3. FTIR deconvolution spectrum of gelatin.
C O
Alanine Cytosine Glycine Tryptophan Phenyl alanine Asparagine Aspartic Glutamine Histidine Leucine Methionine Proline Valine
1851.4 1818.9 1884.0 1871.3 1833.8 1783.3 1822.0 1802.5 1855.1 1869.3 1854.5 1830.8 1841.7
C O
1867.4 1875.9 1869.8
N–H
N–H
3500.3 3607.3 3528.4 3507.5 3487.2 3601.8 3494.1 3508.8 3501.1 3503.9 3503.9
3486.2 3602.6
3518.6
500
Wavenumber [Cm-1]
0,0 4000
Vibration wavelength
500
Accordingly, the deconvolution of the gelatin molecule shows the presence of both aliphatic and aromatic amino acids. However, the peak intensities of the deconvoluted spectra of gelatin show that the percentage of aliphatic amino acids is higher than aromatic amino acids. FTIR spectra of the gelatin give the same information for the main source alpha helix. 4. Conclusion In the present study DFT proved to be a powerful tool for studying protein structure. Detailed molecular structure of gelatin could be assigned using B3LYP/6-31g** level of theory. It is concluded that, the calculated spectrum of each amino acid solves the experimental difficulties of the identification of natural protein. Furthermore, correlating both optimized and vibrational characteristics of NH2 group, the NH bond length draw an inverse relationship with the symmetric frequencies of NH. Whereas the HNH bond angle appear to be increasing with increasing frequency. Moreover, the C O bond length shows an inverse relationship with frequency, while, the OCO angle show increase with frequency. The calculated total dipole moment show direct relationship with C O stretching frequency except for cytosine and aspartic which seems to have higher dipole moment values and glycine which shows lower dipole moment regarding the arrangements of the frequencies. Finally the band gap energy increases with stretching frequency except for cytosine, aspartic and tryptophan. References [1] R.M. Lewis, K.M. Godfrey, A.A. Jackson, I.T. Cameron, M.A. Hanson, The Journal of Clinical Endocrinology and Metabolism 90 (3) (2005) 1594–1598. [2] E.J. Taylor, H.M.R. Hott, K.E. Earle, The Journal of Nutrition 124 (12) (1994) 2555S–2558S. [3] P.K.-L. Brehm, K. Schaumburg, J.S. Johansen, E. Falch, D.R. Curtis, Journal of Medicinal Chemistry 29 (2) (1986) 224–229. [4] J.B. Jordan, P.L. Easton, J.F. Hinton, Biophysical Journal 88 (1) (2005) 224–234. [5] P. Felig, Metabolism 22 (2) (1973) 179–207. [6] J.V. Hughes, T.C. Johnson, Journal of Neurochemistry 26 (1976) 110–113. [7] D. Li, X. Gu, L. Lu, L. Liang, Molecular and Cellular Biochemistry 339 (1–2) (2010) 1–7. [8] J.L. Caulfield, J.S. Wishnok, S.R. Tannenbaum, Journal of Biological Chemistry 273 (21) (1998) 12689–12695. [9] S.-I. Yonekura, N. Nakamura, S. Yoneian, Q.-M. Zhang-Akiyama, Journal of Radiation Research 50 (1) (2009) 19–26. [10] I.K. Battisha, H.H. Afify, M. Ibrahim, Journal of Magnetism and Magnetic Materials 306 (2) (2006) 211–217. [11] M. Ibrahim, E. Koglin, Acta Chimica Slovenica 52 (4) (2005) 159–163. [12] M. Ibrahim, M. Abd-El-Aal, International Journal of Environment and Pollution 35 (1) (2008) 99–110. [13] H.S. Ibrahim, M.A. Ibrahim, F.A. Samhan, Journal of Hazard Mater 168 (2–3) (2009) 1012–1016. [14] M. Ibrahim, A.J. Hameed, A. Jalbout, Applied Spectroscopy 62 (3) (2008) 306–311. [15] M. Ibrahim, Spectrochimica Acta Part A 77 (2010) 1034–1038.
M. Ibrahim et al. / Spectrochimica Acta Part A 81 (2011) 724–729 [16] M. Ibrahim, A.-A. Mahmoud, O. Osman, A. Refaat, El.-M. El-Sayed, Spectrochimica Acta Part A 77 (2010) 802–806. [17] M. Ibrahim, A. Nada, D.-El. Kamal, Indian Journal of Pure and Applied Physics 43 (2005) 911–917. [18] M. Ibrahim, A.-A. Mahmoud, Journal of Comput. Theor. Nanosci 6 (2009) 1523–1526. [19] M. Ibrahim, A. Al-Hossain, Z. Al-Fifi, Journal of Computational and Theoretical Nanoscience 10 (2010) 2240–2244. [20] J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, J.A. Montgomery Jr., T. Vreven, K.N. Kudin, J.C. Burant, J.M. Millam, S.S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G.A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J.E. Knox, H.P. Hratchian,
[21] [22] [23] [24]
729
J.B. Cross, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, P.Y. Ayala, K. Morokuma, G.A. Voth, P. Salvador, J.J. Dannenberg, V.G. Zakrzewski, S. Dapprich, A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B. Foresman, J.V. Ortiz, Q. Cui, A.G. Baboul, S. Clifford, J. Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng, A. Nanayakkara, M. Challacombe, P.M. W. Gill, B. Johnson, W. Chen, M.W. Wong, C. Gonzalez, J.A. Pople, Gaussian, Inc., Wallingford CT, 2004. A.D. Becke, Journal of Chemical Physics 98 (1993) 5648–5652. C. Lee, W. Yang, R.G. Parr, Physical Review B 37 (1988) 785–789. J.P. Perdew, Y. Wang, Physical Review B 33 (1986) 8800–8802. R. Yin, Y. Huang, C. Huang, Y. Tong, N. Tian, Materials Letters 63 (2009) 1335–1337.
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Molecular spectroscopic analyses of gelatin Medhat Ibrahim ∗ , Abdel Aziz Mahmoud, Osama Osman, Mohamed Abd El-Aal, May Eid Spectroscopy Department, National Research Center, 12311, Dokki, Cairo, Egypt
a r t i c l e
i n f o
Article history: Received 24 April 2011 Received in revised form 30 June 2011 Accepted 3 July 2011 Keywords: Natural protein Amino acids FTIR DFT
a b s t r a c t The molecular structure of gelatin was studied using Fourier transform infrared spectroscopy FTIR. The spectrum is subjected to deconvolution in order to elucidate the constituents of the molecular structure. B3LYP/6-31g** was used to study 13 amino acids then the scaled spectrum was compared to those of protein in order to describe the contribution of each amino acid into protein structure. A special interest was paid to the NH and C O region. The reactivity of each amino acid was studied in terms of some important physical parameters like total dipole moment and HOMO/LUMO which describe the interaction of amino acid with their surrounding molecules. Results indicated that B3LYP/6-31g** model is a suitable and precise method for studying molecular structure of protein. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Amino acids are the building units of protein. Among the twenty naturally occurring protein is glycine. Glycine is the simplest amino acid, with no side chain. Therefore, glycine is of particular importance as a structural unit in some proteins like collagens, and in many metabolic pathways acting as a source of one-carbon units. In mammals, collagen contains over 30% glycine, and collagens make up 20–25% of the body’s protein [1]. Glycine is also essential as a metabolic precursor. Readily converted to serine and is involved in the synthesis of both creatine and glutathione. Additionally, glycine is an integral precursor of uric acid [2]. Moreover, Glycine is the most potent inhibitory neurotransmitter in the spinal cord [3]. Amphipathic residues such as tryptophan (Trp) are believed to play a critical role in the structure and function of integral membrane proteins. It is strongly believed that the common occurrence of Trp residues at the aqueous–lipid interface region of transmembrane channels is indicative of its importance for insertion and stabilization of the membrane channel [4]. Alanine is quantitatively the primary amino acid released by muscle and extracted by the splanchnic bed in post absorptive as well as prolonged fasted man. The hepatic capacity for conversion of alanine to glucose exceeds that of all other amino acids. Insulin inhibits gluconeogenesis by reducing hepatic alanine uptake. In contrast, in diabetes, an increase in hepatic alanine extraction is observed in the face of diminished circulating substrate. In prolonged fasting, diminished alanine release is the mechanism whereby gluconeogenesis is reduced. In circumstances in which alanine is deficient,
∗ Corresponding author. Tel.: +20 122727636; fax: +20 233370931. E-mail addresses: [email protected], [email protected] (M. Ibrahim). 1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2011.07.012
such as pregnancy and ketotic hypoglycemia of infancy, fasting hypoglycemia is accentuated [5]. The non-polar amino acid phenylalanine which is naturally present in breast milk of mammals, has attracted attention since chronic excess of phenylalanine in the blood circulatory system has been shown to be associated with deficiencies in the total myelin content both in the human phenyl ketonuric patient and in experimentally induced hyperphenyl alanemia. Assuming that alterations of macromolecular synthesis may be related to the mental retardation often associated with untreated phenyl ketonuria, several investigators have studied the effects of high levels of phenylalanine on neural protein metabolism [6]. Studies indicated that Phe reduced neurite outgrowth of the cortical neurons and inhibited neuronal survival. Furthermore, previous work has shown that Phe affected BDNF mRNA and protein expression. In addition, Phe inhibited the activity of ERK and Akt [7]. Cytosine is one of the main four bases found in DNA and RNA, derived from pyrimidine, it was found that DNA daily attack by oxygen reactive specious could cause deamination of DNA bases, resulting in a direct damage of DNA. It has been reported that there is a correlation between DNA duplex melting and cytosine deamination, indicating that deamination in double-stranded DNA is inversely related to duplex stability [8,9]. Fourier transform infrared spectroscopy is a powerful technique for elucidating molecular structure. It could be utilized in studying nano-materials [10]; polymers [11]; environmental samples [12–14]; biological interaction [15,16]. Both FTIR and molecular modeling were used to study glycine among other structures [17]. The aim was to follow up the vibrational characteristics of COOH. Later on amide as well as COOH and many functional groups were a topic of modeling work to assess their reactivity [18]. The possible interaction of alkali metals as well as heavy metals with protein was conducted using density functional theory method DFT [19]. Alanine was choosing as a model molecule for this interaction.
M. Ibrahim et al. / Spectrochimica Acta Part A 81 (2011) 724–729
The present work is conducted to study gelatin by FTIR. Some amino acids were studied by B3LYP/6-31g** model. The deconvolution of FTIR was conducted and compared with molecular modeling results to assign the vibrational spectrum of gelatin.
725
2.2. Instrumentations
2. Materials and methods
Jasco FTIR 430 Fourier transform infrared spectrometer was used for recording the obtained IR spectra. Spectra were recorded in a spectral range of 4000–400 cm−1 , resolution of 4 cm−1 and scan speed is 2 mm/s.
2.1. Reagents
2.3. Calculation details
Gelatin from bovine skin, Type B was obtained from Sigma– Aldrich (Steinheim, Germany).
Model molecules represent the following amino acids alanine (Ala), cytosine (Cyc), glycine (Gla), tryptophan (Try), phenyl ala-
Fig. 1. Optimized structures of the studied amino acids.
726
M. Ibrahim et al. / Spectrochimica Acta Part A 81 (2011) 724–729
Fig. 1. (Continued )
nine (Ph.Ala), asparagine (Asparg), aspartic (Asp), glutamine (Glu), histidine (His), leucine (Leuc), methionine (Meth), proline (Prol) and valine (Val) were constructed. All calculations were performed on personal computer using the Gaussian 03 program system [20] Geometries were optimized using density functional theory DFT, hybrid Becke 3-Lee–Yang–Parr (B3LYP) exchange correlation functional [20–23] with 6-31g** basis set. HOMO-LUMO frontier energies and vibrational spectra were calculated at the same level of theory. 3. Results and discussion For each amino acid the geometry is optimized at B3LYP/631g**, then vibrational frequencies were calculated at the same level of theory. The optimized amino acids are indicated in Fig. 1.
3.1. Calculated bond lengths and bond angles Table 1 shows the optimized parameters of the calculated amino acids. It is clear that among the studied amino acids, thirteen of them own only one NH2 group (amide) while asparagines and glutamine have two NH2 groups. Moreover, all the same thirteen amino acids have at least one carboxylic group, except cytosine. Geometrical parameters were represented for both amid groups (NH2 and COOH). The bond length of C O ranged from 1.20 to 1.23 A˚ which is nearly constant for all the studied amino acids. Some amino acids show identical NH bonds such as alanine, cytosine, glycine, tryptophan, phenyl alanine, histidine, methionine and valine, while, ˚ which is not Others show by difference in their NH about 0.01 A, a significant difference. So, the two bonds which consist the amide
M. Ibrahim et al. / Spectrochimica Acta Part A 81 (2011) 724–729
727
Fig. 1. (Continued ).
Table 1 B3LYP/6-31g** calculated bond lengths (Å) bond angles (◦ ) for the studied amino acids. LC Alanine Cytosine Glycine Tryptophan Phenyl alanine Asparagine Aspartic Glutamine Histidine Leucine Methionine Proline Valine
LC
O
1.21 1.22 1.20 1.21 1.21 1.23 1.21 1.22 1.21 1.21 1.21 1.21 1.21
O
1.21 1.21 1.21
NH1
NH2
1.02 1.01 1.01 1.02 1.02 1.01 1.01 1.02 1.02 1.02 1.02
1.02 1.01 1.01 1.02 1.02 1.02 1.02 1.01 1.02 1.01 1.02
1.02
1.02
NH1
OCO
OCO
122.4
1.01 1.01
groups are identical. Regarding the bond angles OCO angle ranged from 120◦ to 124◦ . While HNH angle ranged from 106◦ to 118.8◦ . Although the bonds distances of NH2 groups are identical, their angles are not. 3.2. The total dipole moments and energy gaps The calculated dipole moment can be expressed in terms of vector in three directions X, Y and Z. It is considered as being the measure of asymmetry in the molecule charge distribution. The dipole moment is derived from the first derivative which reflects the static behavior of the molecule. Table 2 indicates the calculated total dipole moment. The results show that the amino acids can be arranged in the following increasing order: Ph.Ala > Prol > Val > Ala > Meth > His > Asparg > Leuc > Glu > Try. Table 2 B3LYP/6-31g** calculated HOMO/LUMO energy (E) as eV, total dipole moment (TDM) as Debye.
Alanine Cytosine Glycine Tryptophan Phenyl alanine Asparagine Aspartic Glutamine Histidine Leucine Methionine Proline Valine
NH2
E
TDM
MPG
6.75 5.33 7.39 5.182 4.25 7.11 6.54 7.06 6 7.22 6.23 6.08 6.67
2.34 6.29 5.58 6.45 1.93 4.73 4.38 6.55 4.65 5.41 2.6 1.85 2.14
C1 C1 C1 C1
1.01 1.01
123.9 123.6 122.2 123.6 124.0 123.5 122.7 123.1 122.8 122.6 122.0
122.4
HNH
HNH
107.1 118.1 107.4 107.0 106.0 106.4 106.4 118.7 108.6 107.1 108.0
118.8 107.4
107.2
The difference between highest occupied molecular orbital HOMO and lowest unoccupied molecular orbital LUMO is calculated. These differences together with total dipole moment reflect the reactivity of a given structure with their surrounding molecule. The same arrangement could be made for the studied amino acids. The calculated band gap energy for the studied amino acids is arranged in the following order: Ph.Ala > Prol > Val > Ala > Meth > Hist > Asparg > Leuc > Glu > Gly.
3.3. Vibrational spectrum of gelatin The FTIR absorption spectrum and band assignment of gelatin is indicated in Fig. 2. Gelatin characteristic bands were described by Ibrahim et al. and Yin et al. [16,24] As seen in Fig. 2, the absorption bands of amide groups were located at 3434 cm−1 (NH stretching), 1638 cm−1 (amide I, C O and CN stretching), 1545.51 cm−1 (amide II) and 1243 cm−1 (amide III). These bands could be assigned to the characteristic bands of gelatin. The band at 2923 cm−1 represents the CH2 asymmetric stretching vibration which is followed by another one at 2853 cm−1 , which corresponds to symmetric stretching vibration. The CH2 bending and wagging vibrations raise two bands at 1452 cm−1 and 1337 cm−1 respectively. Finally the CH3 amide group appears at 1031 cm−1 while the COC band appears at 537 cm−1 . In our calculations we concentrate on both C O and N–H vibrations. The spectra indicate that although the geometrical parameters are identical, the frequencies appear different from each other. C O is ranged from 1738.0 cm−1 to 1884.0 cm−1 , while N–H ranged from 3487.2 cm−1 to 3607.3 m−1 . Theoretically, each band appears as a harp line while experimentally, the band appears broad. In order to demonstrate the contribution of amino acids in
728
M. Ibrahim et al. / Spectrochimica Acta Part A 81 (2011) 724–729 Table 3 B3LYP/6-31g** calculated C O and N–H symmetric frequency.
0,4
0,3
Absorbance
NH CH2
0,2
Amide I Amide II
0,1
CH3 COC Amide III
0,0 4000
3500
3000
2500
2000
1500
1000
Fig. 2. FTIR absorption spectrum of gelatin.
the protein structure, the deconvolution of the experimental spectrum was carried out. Extra assignment could be carried out with the deconvolution of the gelatin spectrum as in Fig. 3. Furthermore a correlation between calculated as well as FTIR spectrum could be found. Regarding the NH stretching in region 4000–3000 cm−1 . There are some NH bands which could be attributed to the presence of tryptophan, cytosine and phenyl alanine respectively. This could be further emphasized with the finger print of aromatic ring around 1000 cm−1 and the CH stretching for the aromatic ring at 3088 cm−1 . Regarding the theoretical results performed using B3LYP/6-31g** level of theory, the calculation of E for the thirteen amino acids (Table 2) show that cytosine is 5.3 eV, tryptophan is 5.2 eV, whereas, phenyl alanine is 4.2 eV. Therefore, these three aromatic compounds show the lowest binding energy values among the 13 studied amino acids. Thus, these three amino acids could be the weak points at which the breaking down of big alpha helix structure (collagen in our case) into the beta sheet (gelatin in our study) could occur (see Table 3). From the above results we can conclude that the two peaks of the NH stretching regions represent one aliphatic and one aromatic amino acid. The error in this method is systematic and could be overcome by scale factor to match both experimental and theoretical data. Results may prove that the two main amino acids in gelatin molecule are tryptophan and glycine. The tryptophan is an aromatic amino acid and the glycine is an aliphatic amino acid.
Absorbance
1,0
0,5
3500
3000
2500
2000
1500
1000
-1
Wavenumber [Cm ] Fig. 3. FTIR deconvolution spectrum of gelatin.
C O
Alanine Cytosine Glycine Tryptophan Phenyl alanine Asparagine Aspartic Glutamine Histidine Leucine Methionine Proline Valine
1851.4 1818.9 1884.0 1871.3 1833.8 1783.3 1822.0 1802.5 1855.1 1869.3 1854.5 1830.8 1841.7
C O
1867.4 1875.9 1869.8
N–H
N–H
3500.3 3607.3 3528.4 3507.5 3487.2 3601.8 3494.1 3508.8 3501.1 3503.9 3503.9
3486.2 3602.6
3518.6
500
Wavenumber [Cm-1]
0,0 4000
Vibration wavelength
500
Accordingly, the deconvolution of the gelatin molecule shows the presence of both aliphatic and aromatic amino acids. However, the peak intensities of the deconvoluted spectra of gelatin show that the percentage of aliphatic amino acids is higher than aromatic amino acids. FTIR spectra of the gelatin give the same information for the main source alpha helix. 4. Conclusion In the present study DFT proved to be a powerful tool for studying protein structure. Detailed molecular structure of gelatin could be assigned using B3LYP/6-31g** level of theory. It is concluded that, the calculated spectrum of each amino acid solves the experimental difficulties of the identification of natural protein. Furthermore, correlating both optimized and vibrational characteristics of NH2 group, the NH bond length draw an inverse relationship with the symmetric frequencies of NH. Whereas the HNH bond angle appear to be increasing with increasing frequency. Moreover, the C O bond length shows an inverse relationship with frequency, while, the OCO angle show increase with frequency. The calculated total dipole moment show direct relationship with C O stretching frequency except for cytosine and aspartic which seems to have higher dipole moment values and glycine which shows lower dipole moment regarding the arrangements of the frequencies. Finally the band gap energy increases with stretching frequency except for cytosine, aspartic and tryptophan. References [1] R.M. Lewis, K.M. Godfrey, A.A. Jackson, I.T. Cameron, M.A. Hanson, The Journal of Clinical Endocrinology and Metabolism 90 (3) (2005) 1594–1598. [2] E.J. Taylor, H.M.R. Hott, K.E. Earle, The Journal of Nutrition 124 (12) (1994) 2555S–2558S. [3] P.K.-L. Brehm, K. Schaumburg, J.S. Johansen, E. Falch, D.R. Curtis, Journal of Medicinal Chemistry 29 (2) (1986) 224–229. [4] J.B. Jordan, P.L. Easton, J.F. Hinton, Biophysical Journal 88 (1) (2005) 224–234. [5] P. Felig, Metabolism 22 (2) (1973) 179–207. [6] J.V. Hughes, T.C. Johnson, Journal of Neurochemistry 26 (1976) 110–113. [7] D. Li, X. Gu, L. Lu, L. Liang, Molecular and Cellular Biochemistry 339 (1–2) (2010) 1–7. [8] J.L. Caulfield, J.S. Wishnok, S.R. Tannenbaum, Journal of Biological Chemistry 273 (21) (1998) 12689–12695. [9] S.-I. Yonekura, N. Nakamura, S. Yoneian, Q.-M. Zhang-Akiyama, Journal of Radiation Research 50 (1) (2009) 19–26. [10] I.K. Battisha, H.H. Afify, M. Ibrahim, Journal of Magnetism and Magnetic Materials 306 (2) (2006) 211–217. [11] M. Ibrahim, E. Koglin, Acta Chimica Slovenica 52 (4) (2005) 159–163. [12] M. Ibrahim, M. Abd-El-Aal, International Journal of Environment and Pollution 35 (1) (2008) 99–110. [13] H.S. Ibrahim, M.A. Ibrahim, F.A. Samhan, Journal of Hazard Mater 168 (2–3) (2009) 1012–1016. [14] M. Ibrahim, A.J. Hameed, A. Jalbout, Applied Spectroscopy 62 (3) (2008) 306–311. [15] M. Ibrahim, Spectrochimica Acta Part A 77 (2010) 1034–1038.
M. Ibrahim et al. / Spectrochimica Acta Part A 81 (2011) 724–729 [16] M. Ibrahim, A.-A. Mahmoud, O. Osman, A. Refaat, El.-M. El-Sayed, Spectrochimica Acta Part A 77 (2010) 802–806. [17] M. Ibrahim, A. Nada, D.-El. Kamal, Indian Journal of Pure and Applied Physics 43 (2005) 911–917. [18] M. Ibrahim, A.-A. Mahmoud, Journal of Comput. Theor. Nanosci 6 (2009) 1523–1526. [19] M. Ibrahim, A. Al-Hossain, Z. Al-Fifi, Journal of Computational and Theoretical Nanoscience 10 (2010) 2240–2244. [20] J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, J.A. Montgomery Jr., T. Vreven, K.N. Kudin, J.C. Burant, J.M. Millam, S.S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G.A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J.E. Knox, H.P. Hratchian,
[21] [22] [23] [24]
729
J.B. Cross, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, P.Y. Ayala, K. Morokuma, G.A. Voth, P. Salvador, J.J. Dannenberg, V.G. Zakrzewski, S. Dapprich, A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B. Foresman, J.V. Ortiz, Q. Cui, A.G. Baboul, S. Clifford, J. Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng, A. Nanayakkara, M. Challacombe, P.M. W. Gill, B. Johnson, W. Chen, M.W. Wong, C. Gonzalez, J.A. Pople, Gaussian, Inc., Wallingford CT, 2004. A.D. Becke, Journal of Chemical Physics 98 (1993) 5648–5652. C. Lee, W. Yang, R.G. Parr, Physical Review B 37 (1988) 785–789. J.P. Perdew, Y. Wang, Physical Review B 33 (1986) 8800–8802. R. Yin, Y. Huang, C. Huang, Y. Tong, N. Tian, Materials Letters 63 (2009) 1335–1337.