Analytica Chimica Acta 444 (2001) 205–210 Resonance light-scattering spectroscopic determination of protein with pyrocatechol violet Xin Cong a , Zhong-Xian Guo b , Xiao-Xia Wang a , Han-Xi Shen a,∗ a Department of Chemistry, Nankai University, Tianjin 300071, China b College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China Received 18 December 2000; received in revised form 11 April 2001; accepted 15 May 2001 Abstract At pH 1.4–2.0 and in the presence of Triton X-100 the resonance light-scattering (RLS) spectrum of pyrocatechol violet (PV) has a maximum peak at 341 nm with two wide bands centering on 399 and 468 nm, respectively. If micro protein coexists in the system, the RLS intensities of PV at 399 and 468 nm are signiﬁcantly increased by protein due to the binding interaction between protein and PV. Based on this, a new assay for protein is proposed. The calibration graphs for bovine and human serum albumin are linear up to 8.0 and 9.0 mg l−1 with 3σ detection limits of 5.2 and 6.9 × 10−5 mg l−1 , respectively. The method is characterized by high sensitivity, short reaction period, simplicity and good stability of the chemical system. Results for human serum, urine and saliva are in agreement with those obtained by the Bradford method with relative standard deviations of 1.5–2.3% (n = 5). © 2001 Elsevier Science B.V. All rights reserved. Keywords: Protein assay; Resonance light-scattering spectroscopy; Pyrocatechol violet 1. Introduction is seriously interfered by coexisting substances while the bromocresol green method is liable to disturbance The quantitative determination of protein is consid- by turbidity and is not sensitive enough. Therefore, a erably essential in biochemistry and clinical medicine. number of assays have been continuously reported in Currently most of the widely used protein assays are recent years, such as those based on spectrophotome- spectrophotometric, such as the Lowry , Bradford tric [9–12], ﬂuorometric [13–17], chemiluminesence [2,3], bromophenol blue [4,5] and bromocresol green  and electrochemical . [6,7]. However, they all have some limitations in Since its ﬁrst introduction to the quantitative de- terms of sensitivity, selectivity, stability and simplic- termination of biomacromolecules in 1996 [20,21], ity. For example, the Lowry procedure is complicated resonance light-scattering (RLS) technique has grad- and subject to interference by coexisting substance ually gained regards from analytical chemists [22,23]. such as thiol reagents, some metal ions and amino It is characterized by high sensitivity, convenience acids . The bromophenol blue method has low in performance and simplicity in apparatus (usually sensitivity with dynamic range of 10–80 mg l−1 and common spectroﬂuorometer). By using RLS spec- troscopy, organic dye probes used in spectrophotomet- ∗ Corresponding author. Tel.: +86-22-23502458; ric assays for protein and nucleic acids can become fax: +86-22-23501705. much more sensitive. For instance, the dynamic E-mail address: email@example.com (H.-X. Shen). range for bovine serum albumin (BSA) of the RLS 0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 1 ) 0 1 1 2 6 - 6 206 X. Cong et al. / Analytica Chimica Acta 444 (2001) 205–210 method with bromophenol blue was reported to be prepared by mixing 50 ml of 1 mol l−1 hydrochloric 0.34–18.7 mg l−1 while the spectrophotometric pro- acid and 53.5 ml of 1.0 mol l−1 sodium acetate, dilut- cedure can be used just for the protein concentrations ing the solution to 250 ml with water, and adjusting to greater than 10 mg l−1 . It is also true for ethyl pH 1.8. A 2.0% (v/v) Triton X-100 solution was ob- violet in the determination of DNA . Up-to-date tained by dissolving 2.0 ml of Triton X-100 (Sigma) several RLS procedures have been prescribed for and diluting to 100 ml with water. The working solu- protein assays using organic dye [21,25–28] or metal tion of Coomassie brilliant blue G-250 (CBB G-250, complex . Fluka) was prepared by the recommended procedure Pyrocatechol violet PV, a very usual dye, was used . Unless otherwise mentioned all chemicals were in its complex form with molybdenum  or tin of analytical grade or the best grade commercially  for spectrophotometric determination of protein. available. Deionized water was used throughout. However, PV itself has little practical usefulness in protein spectrophotometry due to poor sensitivity. 2.3. Procedure Here, PV is found a highly sensitive RLS probe for protein and a simple and practical method for pro- Into a 10 ml colorimetric tube, 1.0 ml of 2.0% Tri- tein quantiﬁcation is described on the basis of the ton X-100, 1.0 ml of Walpole buffer (pH 1.8), 0.30 ml enhancement effect on the RLS intensity of PV in the of 1.0 × 10−3 mol l−1 PV solution and an appropriate presence of Triton X-100. volume of sample or protein working solution were placed in turn. The mixture was diluted to 10 ml with water and mixed thoroughly. The RLS spectrum was 2. Experimental obtained by synchronously scanning the excitation and emission monochromators ( λ = 0.0 nm) of 2.1. Apparatus RF-540 spectroﬂuorometer in the wavelength region from 300 to 500 nm. The excitation and emission slits RLS intensities were measured with 10 mm quartz were set to 2.0 nm. The RLS intensity was measured cells on a Shimadzu RF-510 spectroﬂuorometer at the maximum wavelength (399 nm) with RF-510 (Kyoto, Japan). RLS spectra were obtained with a spectroﬂuorometer. The enhancement of the RLS in- Shimadzu RF-540 spectroﬂuorometer. pH values were tensity ( I) is the difference in intensities between measured with a pH S-2 acidimeter (Shanghai Second sample (I) and blank (I0 ). Analytical Instrument Plant, Shanghai, China). 2.2. Reagents 3. Results and discussion Appropriate amounts of human serum albumin 3.1. Absorption and resonance (HSA) and BSA (Sino-American Biotechnology, light-scattering spectra Tianjin, China) and cytochrome c from yeast (Cyt c, Shanghai Lizhu Dongfeng Biotechnology, Shanghai, Based on the reaction of protein, PV and metal China) were directly dissolved in water to prepare ions including molybdenium and tin in the presence stock solutions and stored at 0–5◦ C. The working so- of surfactant, Fujita et al. [30,31] described two spec- lutions were obtained by diluting the stock solutions trophotometric procedures for the protein determina- with water just prior to use. A 386.4 mg mass of PV tion. According to their discussion, protein reacts with purchased from Tianjin First Chemicals Plant, Tianjin, metal–PV complex to develop a ternary complex. China, was dissolved and diluted to 100 ml with wa- Although the interaction of PV and protein occurs at ter to prepare a 1.0 × 10−2 mol l−1 solution. When acidic media due to the electrostatic binding, it has stored in a brown ﬂask or in the dark the solution is not been proposed for protein spectrophotometric de- stable for several months. A PV working solution of termination yet. The main reason can be seen from 1.0 × 10−3 mol l−1 was prepared by diluting the stock the absorption spectra of PV in the absence and pres- solution with water. A pH 1.8 Walpole buffer was ence of protein as shown in Fig. 1. The absorption X. Cong et al. / Analytica Chimica Acta 444 (2001) 205–210 207 Fig. 1. Absorption spectra against water of (a) 1.0 × 10−4 mol l−1 PV; (b) 8.0 mg l−1 BSA and (c) 1.0 × 10−4 mol l−1 PV-8.0 mg l−1 BSA at pH 1.8 and in the presence of 0.2% Triton X-100. Fig. 2. Resonance light-scattering spectra of (a) PV; (b) 2.0 mg l−1 BSA, (c) 4.0 mg l−1 BSA; (d) PV-2.0 mg l−1 BSA and (e) PV- difference between such two situations is too small to BSA in the presence of 0.2% Triton X-100 and at pH 1.8. All the explore a relatively sensitive procedure. However, we concentration of PV is 3.0 × 10−5 mol −1 . can ﬁnd that the case is different for RLS spectrum and intensity. Fig. 2 shows that the RLS spectrum of PV has a isoelectric points of BSA, HSA and cytochrome c are sharp and maximum peak at 341 nm and two wide 4.6–4.7, 4.8 and >10, respectively . The dissoci- bands centering on 399 and 468 nm, respectively, with ated sulfonic group in PV can interact with positively relatively small intensity. When protein coexists in charged groups in proteins, such as protonated amino the system, the RLS spectrum of PV has a signiﬁcant group. This makes PV and protein bind through elec- change. The peak at 341 nm disappears, and that at trostatic forces. Meanwhile, the aromatic rings of PV 468 nm becomes sharp. Such a phenomenon of this and protein have interaction due to non-electrostatic system is different from those in reported systems. The effects, such as hydrophobic and Van der Waals forces. RLS intensity of PV is greatly enhanced by the pres- Therefore, many PV dye molecules can position at ence of BSA in the examined range 300–500 nm, and the non-speciﬁc binding sites of a protein molecule, maximum enhancement is present around 399 nm. On which leads to the aggregation of PV molecules on the other hand, when PV is absent the difference of the surface of protein molecule. The aggregation RLS intensity between various BSA concentrations is will produce relatively large particles, and result in very small. Therefore, the enhancement effect of pro- the enhancement effect of protein on RLS intensity tein can serve as a basis for protein assay. of PV. Such an enhancement effect of protein on RLS in- tensity of PV can be understood in view of molecular 3.2. Optimization of reaction conditions structure. PV is an acid dye with triphenylmethane structure. A PV molecule has hydrophilic groups, In weakly acidic medium PV, a negatively charged i.e. a sulfonic and 3-phenol hydroxyl groups, and a dye binds with positively charged protein, and the planar and hydrophobic triphenylmethane ring. At reaction is obviously affected by acidity. As Fig. 3 pH 1.8, PV and protein are negatively and positively shows, the enhancement of RLS intensity of PV by charged, respectively, since pKal of PV is <1  and BSA reaches a maximum and remains constant in 208 X. Cong et al. / Analytica Chimica Acta 444 (2001) 205–210 Fig. 3. Impact of pH on enhancement on RLS intensity of Fig. 4. Effect of PV concentration on RLS intensity enhancement 3.0 × 10−5 mol l−1 PV by 4.0 mg l−1 BSA in the presence of 0.2% by 4.0 mg l−1 BSA in the presence of 0.2% Triton X-100. Triton X-100. the pH range 1.4–2.0. So pH 1.8 is recommended. chain groups in molecules. Among examined nonionic Furthermore, the effect of buffer nature on I was ex- and polymeric surfactants, Triton X-100 has the best amined, and several kinds of buffers, such as Walpole, enhancing effect, as shown in Table 1. Further in- potassium chloride–HCl and glycine–HCl, gave the vestigation showed that a suitable volume of 2.0% is almost same result. Walpole buffer of pH 1.8 was 0.6–2.0 ml; 1.0 ml was used subsequently. A reason- chosen, and in a ﬁnal 10 ml solution, 1.0 ml of this able explanation of these phenomena is assumed to be buffer was suitable. the binding process between protein and Triton X-100 PV concentration affects the RLS intensity en- by Van der Waals forces and hydrophobic interactions. hancement of the system. When PV concentration is As shown in Table 2, the addition order of reagents too low, a speciﬁed amount of protein cannot interact affects the enhancement of protein on the RLS inten- fully with PV, in other word, limited PV molecules sity. The best order is ﬁnal addition of protein (or PV) cannot occupy all non-speciﬁc binding sites of protein after the addition and mixing of Triton X-100, buffer coexisting in the system. On the other hand, the rel- and PV (or protein). The reason for this phenomenon atively strong absorption of highly concentrated PV is not clear yet and worthy of further study. weakens the RLS intensity of the system. As shown in Fig. 4, the enhancement of protein on RLS inten- sity of PV is small when PV concentration is very Table 1 low. It increases with the increasing of PV concentra- Inﬂuence of surfactant on enhancement effect of 4.0 mg l−1 BSA on the RLS intensity of 3.0 × 10−5 mol l−1 PV tion, and reaches a maximum and remains constant when PV concentration is in the range 2.8 × 10−5 Surfactant (concentration) λmax (nm) I to 3.5 × 10−5 mol l−1 . Thus, a PV concentration of Polyvinyl alcohol (0.2% m/v) 387 31.6 3.0 × 10−5 mol l−1 was chosen. Tween 80 (0.2% v/v) 399 19.4 The experiments showed that surfactant has an ef- Tween 20 (0.2% v/v) 399 17.9 Emulsiﬁer OP (0.2% v/v) 399 23.7 fect on the system. Cationic and anionic surfactants Triton X-100 (0.2% v/v) 399 44.1 interfere with the determination due to charged long X. Cong et al. / Analytica Chimica Acta 444 (2001) 205–210 209 Table 2 3σ limits of detection for BSA and HSA are 5.2 and Inﬂuence of the addition order of reagents on the RLS enhancement 6.9 × 10−5 mg l−5 , respectively (here σ represents by BSA the standard deviation of 11 blank measurements). Addition order of reagents I This procedure can be used for quantiﬁcation of cy- Triton X-100, buffer, PV, BSA 44.7 tochrome c with a linear range up to 9.0 mg l−1 and Triton X-100, buffer, BSA, PV 44.3 detection limit of 5.8 × 10−5 mg l−1 . The linear re- PV, buffer, Triton X-100, BSA 44.1 gression equation for cytochrome c is as follows: PV, BSA, Triton X-100, buffer 12.8 I = 0.825 + 11.9 C (mg l−1 ) with a correlation PV, Triton X-100, BSA, buffer 34.6 Triton X-100, PV; BSA, buffer 35.8 coefﬁcient of 0.9994 (n = 10). From the dynamic buffer, PV, BSA, Triton X-100 11.5 ranges and detection limits for serum albumins, it is very clear that this method is much more sensitive than most of currently used and recently prescribed spectrophotometric [1–12] and ﬂuorometric  pro- After the dilution of the mixture of protein and cedures, and at least comparably sensitive with most reagents, the RLS intensity of the system reaches a of RLS methods [21,25–28]. maximum within 5 min and remains constant for at least 3.0 h. An incubation time of 10 min was used in 3.4. Effect of coexisting substances the study. Therefore, the system allows ample time to measure the RLS intensity enhancements of a great Under the optimum conditions various non-protein number of samples. coexisting substances were examined for interfer- ence. For 4.0 mg l−1 BSA, the following metal ions 3.3. Calibration curves and sensitivity and amino acids at given concentrations in mol l−1 and mg l−1 , respectively, caused a recovery within The calibration graphs for BSA, HSA and cy- 94.0–105.6%: Ca2+ , 5.0 × 10−5 ; Cd2+ , 3.0 × 10−5 tochrome c were constructed under the optimized Co2+ , 3.0 × 10−5 Cu2+ , 5.0 × 10−5 Fe3+ , 5.0 × 10−5 conditions. The linear range for BSA is 0–8.0 mg l−1 Hg2+ , 1.0×10−5 Mg2+ , 5.0×10−5 Mn2+ , 5.0×10−5 ; and the linear regression equation is as follows: Ni2+ , 5.0×10−5 ; Pb2+ , 1.0×10−5 ; Zn2+ , 1.0×10−5 ; I = −2.24 + 11.7 C (mg l−1 ), with a correlation l-Val, 20; l-His, 40; l-Cys, 40; l-Lys, 20; l-Arg, coefﬁcient of 0.9994 (n = 9). The dynamic range 40; l-Try, 20; l-Pro, 50; l-Gln, 50; l-Ser, 40; l-Leu, for HSA is 0–9.0 mg l−1 and the linear regression 40; l-Asp, 40; l-Tyr, 20; Gly, 50. The presence of equation is as follows: I = 3.72 + 10.9 C (mg l−1 ) glucose, sodium dodecylsulfate and cetyltrimethy- with a correlation coefﬁcient of 0.9997 (n = 10). The lammonium bromide at 20, 18 and 18 mg l−1 resulted Table 3 Determination results of human ﬂuids samples (n = 5) Sample This method CBB assay t-testa (t) Foundb RSD(%)c Foundb RSD(%)c Serum 1 73.2 ± 2.1 2.3 74.5 ± 1.9 2.1 1.27 Serum 2 68.8 ± 1.5 1.8 69.1 ± 1.2 1.4 0.47 Urine 1 48.9 ± 1.3 2.1 50.0 ± 1.2 2.0 1.72 Urine 2 53.3 ± 1.5 2.3 53.9 ± 1.4 2.1 0.80 Saliva 1 684 ± 13 1.5 689 ± 2.1 2.5 0.56 Saliva 2 779 ± 18 1.9 790 ± 24 2.4 1.02 a t0.95,8 = 2.31, t0.90,8 = 1.86. √ b ¯ ¯ The found result is expressed as x ± (tP ,n−1 S/ n), where tP ,n−1 = t0.95,4 = 2.78, and x and s are the mean and standard deviation, respectively. The protein concentration in serum is expressed in mg ml−1 , and that in other samples is in mg l−1 . c Relative standard deviation. 210 X. Cong et al. / Analytica Chimica Acta 444 (2001) 205–210 in a recovery of 97.4, 94.7 and 90.4%, respectively. References Glucose, amino acids and metal ions, able to complex with PV, have relatively high tolerable limits. There-  O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. fore, compared with common dye-binding methods, Biol. Chem. 193 (1951) 265.  M.M. Bradford, Anal. Biochem. 72 (1976) 248. the present procedure is selective.  T. Zor, Z. Selinger, Anal. Biochem. 236 (1996) 302.  R. Flores, Anal. Biochem. 88 (1978) 605. 3.5. Sample determination  K. Jung, E. Nickel, M. Pergande, Clin. Chim. Acta 187 (1990) 163.  F.L. Rodkey, Clin. Chem. 11 (1965) 478. The present method was applied to quantify total  B.T. Doumas, W.A. Watson, H.G. Biggs, Clin. Chim. Acta protein in human body ﬂuids including serum, urine 31 (1971) 87. and saliva. Human serum samples, obtained from the  G.L. Peterson, Anal. Biochem. 100 (1979) 201. Nankai University Hospital, were stored at 0–5◦ C  H.S. Soedjak, Anal. Biochem. 220 (1994) 142. and diluted 1000-fold with deionized water just be-  A.A. Waheed, P.D. Gupta, Anal. Biochem. 233 (1996) 249.  K. Zhu, K.A. Li, S.Y. Tong, Anal. Lett. 29 (1996) 575. fore the determination. Human saliva samples were  Z.X. Guo, Y.M. Hao, X. Cong, H.X. Shen, Anal. Chim. Acta diluted 10-fold to become sample’s working solu- 403 (2000) 225. tions. Fresh human urine samples were readily used  J. Nakamura, S. Igarashi, Anal. Lett. 29 (1996) 981. as working solutions for protein assay. Table 3 sum-  N. Li, K.A. Li, S.Y. Tong, Anal. Biochem. 233 (1996) 151. marizes the results, which are very close to those, ob-  J. Yuan, K. Matsumoto, J. Biomed. Anal. 15 (1997) 1397.  M.A. Kessler, A. Meinitzer, O.S. Wolfbeis, Anal. Biochem. tained by the Bradford method. The t-test estimation 248 (1997) 180. showed there are no signiﬁcant differences between  D.H. Li, H.H. Yang, H. Zhen, Y. Fang, Q.Z. Zhu, J.G. Xu, the results obtained by the proposed method and Anal. Chim. Acta 401 (1999) 185. Bradford assay at the conﬁdence probability of 0.95  K. Tsukagoshi, Y. Okumura, H. Akasaka, R. Nakajima, T. or 0.90. Hara, Anal. Sci. 12 (1996) 525.  H.M. Zhang, Z.W. Zhu, N.Q. Li, Fresenius’ J. Anal. Chem. 363 (1999) 408.  C.Z. Huang, K.A. Li, S.Y. Tong, Anal. Chem. 68 (1996) 2259. 4. Conclusions  C.Q. Ma, K.A. Li, S.Y. Tong, Anal. Biochem. 239 (1996) 86.  Y.F. Li, C.Z. Huang, X.L. Hu, Chinese J. Anal. Chem. 26 (1998) 1508. The new protein assay described here is based on  Y.T. Wang, K.A. Li, S.Y. Tong, Chem. J. Chinese Univ. 21 the interaction of protein and PV, a very cheap and (2000) 1491. commercially available dye, and utilizes the reso-  T.J. Li, H.X. Shen, Chem. J. Chinese Univ. 19 (1998) 1570. nance light-scattering technique. The enhancement  C.Z. Huang, K.A. Li, S.Y. Tong, Anal. Sci. 13 (1997) 263. effect of Triton X-100 is also involved. The method  C.Z. Huang, Y.F. Li, J.G. Mao, D.G. Tan, Analyst 123 (1998) 1401. is highly sensitive, reproducible, relatively free from  Q.F. Li, X.G. Chen, H.Y. Zhang, C.X. Xue, S.H. Liu, Z.D. interference, with reaction rapidity and good sta- Hu, Analyst 125 (2000) 1483. bility. Therefore, it is advantageous over most of  Z.X. Guo, H.X. Shen, Anal. Chim. Acta 408 (2000) 177. existing protein assays. It has been used in the detec-  Z.X. Guo, H.X. Shen, Spectrochim. Acta A 55 (1999) tion of protein in human ﬂuids yielding satisfactory 2919.  Y. Fujita, I. Mori, S. Kitano, Chem. Pharm. Bull. 32 (1984) results. 4161.  Y. Fujita, I. Mon, T. Matsuo, Bunseki Kagaku 44 (1995) 733.  Y.E. Zhang, H.S. Zhang, Z.H. Chen, Handbook of Modern Acknowledgements Chemical Reagents, Part IV (Chromogenic Reagents for Inorganic Ions), Chemical Industry Press, Beijing, PR China, 1989, p. 340. The authors thank gratefully the National Natural  P.X. Tang, Z.J. Tao, O.R. Qi, J.J. Xu, Handbook of Modern Science Foundation of China for its ﬁnancial aid for Chemical Reagents, Part 1 (Biochemical Reagents), Chemical the project (Grant No. 29875011). Industry Press, Beijing, PR Chian, 1990, pp. 262–422.
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