Abstract Under acidic conditions, the reaction between3,5,7,2′,4′-pentahydroxyflavone (morin) and proteins en-hances the weak light scattering of morin drastically. Theenhanced light scattering intensity is proportional to thecontent of proteins. This fact is the basis of a new methodfor the quantitative analysis of proteins. The linear rangeis 0.45–7.15 and 0.46–11.14 µg/mL for BSA and HSA,respectively. The relative standard deviation is 3.76% (n =12) for BSA in the middle of the linear range. The resultsof this assay for human serum samples were comparablewith those from the traditional method (CBB method).There is almost no interfere from amino acids and most ofthe metal ions. The scattering spectrum of morin was alsodiscussed.

Introduction

Protein determination is always important in clinical ap-plications. The traditional methods are the Lowry method[1, 2] and the Bradford method [3, 4]. Some spectropho-tometric methods [5–8], fluorometric methods [9, 10] anda chemiluminescence method [11] were also developed.Nevertheless, searching for a sensitive spectrophotometricprobe for protein structure studies and analyses is still anactive field for chemists and biochemists. Pasterneck et al.[12, 13] studied porphyrin associates with DNA and theaggregation of chlorophyll by resonance light scatteringtechnique, and pointed out that light scattering techniqueis very sensitive for probing the aggregation of macro-molecules. Huang et al. [14] firstly used the resonancelight scattering technique to establish a new sensitivespectrophotometric method for DNA determination.Later, Ma et al. [15–18] used this technique for protein as-say. In this paper, a new method for protein assay by

Rayleigh light scattering (RLS) with morin is developed.This method is simple, sensitive and rapid for protein de-termination.

Morin (3,5,7,2′,4′-pentahydroxyflavone), a kind of O, O-donating chelating reagent, is one of the polyhydroxy-flavones most frequently used as analytical reagent, espe-cially as an fluorescent reagent, and its structure is shownas follows:

Experimental

Apparatus. A Shimadzu Model RF-540 fluorimeter (Kyoto, Japan)was used to obtain RLS intensity and spectra by scanning with thesame excitation and emission wavelengths. A quartz cell (1 × 1 cmcross-section) was used for detection. The slit-widths for both ex-citation and emission are 5 nm. The pH values were measured witha Model pH-3 meter (Shanghai, China). A Shimadzu Model UV-265 recording spectrophotometer (Japan) was used for recordingthe absorption spectra.

Reagents. Morin was purchased from E. Merk company (Ger-many). Protamine sulfate, bovine serum album (BSA), humanserum album (HSA), hemoglobin bovine, γ-globulin (γ-G) andlysozyme were purchased from Sigma. Chymotrypsin was ob-tained from the Institute of Biochemistry (Academia Sinica). Al-bumin (chicken egg) and pepsin were provided by the ShanghaiBiochemical Institute (China). The protein concentrations were de-termined by spectrophotometry at 280 nm using ε1% values as fol-lows: BSA, 6.6 [19, 20]; lysozyme, 26.04 [20]; HSA, 5.3 [19, 21];cytochrome c, 17.1 [22, 23]; egg albumin, 7.5 [20]; γ-G, 13.8 [20].The concentrations of hemoglobin, protamine sulfate, pepsin andα-chymotrypsin were determined as follows [24]: protein concen-tration (µg/mL) = 144 × (A215-A225). A215 and A225 are the ab-sorbance at 215 nm or 225 nm, respectively, measured using a cellof 1 cm.

All other chemicals were of analytical grade or the best gradecommercially available. Morin solution (0.09%) was preparedwith ethanol, and used as working solution. Clark-Lubs buffer (pH

Ya-Ting Wang · Feng-Lin Zhao · Ke-An Li ·Shen-Yang Tong

Microdetermination of proteins by enhanced Rayleigh light scattering spectroscopy with morin

Fresenius J Anal Chem (1999) 364 :560–564 © Springer-Verlag 1999

Received: 11 October 1998 / Revised: 18 February 1999 / Accepted: 24 February 1999

ORIGINAL PAPER

Ya-Ting Wang · Feng-Lin Zhao · Ke-An Li (Y) ·Shen-Yang TongCollege of Chemistry and Molecular Engineering, Peking University, Beijing 100871, P.R. China

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4.2) was used to control the pH of the reaction system. Fresh hu-man plasma was purchased from the Hospital of Peking Universityand centrifuged, then the upper solution was separated and diluted2000-fold with water.

General procedure. In most experiments, 1.0 mL of morin solution(0.09%), an appropriate volume of sample or standard solution ofBSA and 2.0 mL of buffer were mixed and diluted to 10.0 mL withwater. Light scattering spectra measurements were performed ac-cording to Pasternack et al. [12], i.e., the spectra were obtained byscanning synchronously at the same excitation and emission wave-lengths in the range of 300–700 nm. For RLS intensity measure-ments, the excitation and emission wavelengths were kept at 470 nm.

Results and discussion

Reaction and spectral characteristics

This reaction between morin and proteins is rapid andonly needs a few minutes. The signal is stable for at least2 h. The mixing sequence of reagents affects the RLS in-tensity. Three kinds of mixing sequence were investi-gated. In the first sequence, buffer was added to morin so-lution, then BSA was added and diluted to 10 mL withwater. In the second, BSA was added to morin solutionand buffer was added as the last reagent. And in the third,buffer was added to BSA solution and morin was addedfinally. Among the three sequences, mixing morin andBSA previously and then adding buffer solution will re-sult in higher RLS responses, so this sequence was se-lected generally.

The spectra of light scattering, transmission and ab-sorption are presented in Fig.1. It can be seen that thepeak of the light scattering (LS) spectra of morin is situ-ated at the wavelength of maximum transmission ofmorin, in other words, the peak of the LS spectra appearedat the wavelength of minimum absorption of morin. Thereare two reasons for this result. Not only does absorptiondecrease the scattering intensity, but also the thermal lens-ing caused by absorption results in a large decrease in theobserved intensity due to beam defocusing [25].

The basic theory for RLS has been described previ-ously [12, 25–27]. Typical resonance enhanced Rayleighlight scattering bands are expected for large aggregates atwavelengths where the molar absorbance of the associateis large, such as porphyrin [12], in which the RLS peakappeared within the Soret band of absorption spectrum. Inthis paper, the peak of light scattering of morin does notappear within the envelope of the absorption spectrum ofmorin. The possible reasons are as follows: usually theenhanced resonance Rayleigh scattering appeared at alonger wavelength near the absorption peak [25, 27],however the intensity of enhanced RLS within the absorp-tion region was decreased by the absorption. The size ofmorin and morin-BSA particles may not be larger thanthat of porphyrin and porphyrin-DNA due to differentmolecular weight. Because Isca = k1v2 and Iabs = k2v (whereIsca and Iabs is the intensity of scattering and absorptionlight, respectively, v is the size of the scattering particle,both k1 and k2 are the constants) [12, 28], the RLS inten-sity for porphyrin or chlorophyll is high enough to over-

come the absorption loss. On the other hand, porphyrinhas a sharply rising edge on the absorption band as thediphenylpolyene [29]. Therefore, porphyrin and por-phyrin-DNA show a characteristic resonance enhancedRayleigh light scattering but morin and morin-BSA donot.

The present assay is carried out within the non-absorb-ing range of morin, so the reaction system could betreated as transparent solution. Therefore, the quantitativebasis for this assay is in accordance with the Rayleigh for-mula [30]:

Rθ = 9π2 N0 v2 (1 + cos2 θ)[(n12 –n2

2)/(n12 +2n2

2)]2/2λ4

Where Rθ is the Rayleigh ratio at 90° scattering angle(Rayleigh ratio describes the scattering ability of the sys-tem), n1 and n2 is the refractive index of the solute andmedium respectively, λ is the wavelength, ν is the size ofthe scattering particles, N0 is the number of particles perunit. When the species of the system is fixed, then ν isconstant. Because the scattering particle is an electrostaticmorin-protein complex, the number of scattering particleis determined by the concentration of protein added at afixed concentration of morin. Therefore N0 is proportionalto the concentration of the protein added, i.e. at fixed ex-perimental conditions, the scattering light intensity I isonly determined by the protein concentration C.

I = k · C

Optimization of the general procedure

Figure 2 shows that the pH affects the RLS intensity seri-ously, due to the dissociation and ion charge of morin and

Fig.1 Absorption, Transmission and Rayleigh light scatteringspectra a at pH 4.20: absorption spectrum (––––), transmission (- - - - -) and Rayleigh light scattering spectrum (– – – –) for morin.The curve (– · – · –) represents the RLS spectra of morin-BSA. a: Absorption, Transmission and Rayleigh light scattering spectrahave different units for easy comparison

BSA. The optimized pH is found to be 4.0 ~ 4.7 and con-trolled with pH 4.2 Clark-Lubs buffer. At this pH, BSAexists in cationic form (pI of BSA is 4.8), and morin afterdissociation of protons as anion with 1 to 2 charges. Theseions can form ion-pair associates by electrostatic forces.The acid-base equilibrium of morin is shown as follow[31]:

Η5L ←→pk1 = 1 H4L– ← pk2 = 4.8→ H3L2–←→pk3 = 7 H2L3–←→pk4 = 9

HL4– ← pk5 = 13→ L5–

The effect of the morin concentration on the response ofthe reaction system (for 3.58 µg/mL BSA and pH 4.2)was also investigated. When the morin concentration isless than 0.008%, an increase of morin concentration willenhance the RLS intensity of the morin-BSA system sig-nificantly. However, when the concentration of morin inthe system is more than 0.008%, the signal hardly in-creases. In addition, we found that high concentrations ofmorin produced precipitates causing high errors in the de-termination. In order to get a wider linear range, 0.009%morin solution was used.

Effect of surfactants

From Fig.3 it can be seen, that the surfactants affect thedetermination seriously. With increasing cetyltrimethyl-ammonium bromide (CTAB) concentrations, an enhance-ment of signals of both morin and morin-BSA system isobserved, but for a CTAB concentration above 0.0006%,the signal of the reagent blank is greater than that of themorin-BSA system. This may be explained by the factthat morin reacts with CTAB and forms a morin-CTABassociate [32], and this associate is insoluble in water.Hence the associate particles enhanced the RLS signal ofmorin significantly. On the other hand, CTAB is posi-tively charged, so it will compete with BSA for morin,and this may lead to a weaker signal of the BSA-morinsystem. Addition of sodium dodecyl sulfate (SDS) alsoaffects the signal seriously. The RLS intensity of both, themorin and morin-BSA system, increases strongly, but the

RLS difference between morin and morin-BSA almostkeeps constant. Because addition of Triton X-100 willchange the micro-environment of the morin-BSA system,the RLS difference between morin and morin-BSA in-creases strongly when its concentration is greater than0.0015%. β-Cyclodextrin (β-CD) hardly affects themorin-BSA signal, however, it enhances the signal ofmorin a little bit.

Interfering substances

Amino acids hardly interfere with this assay (see Table 1).The effect of ionic strength (NaCl was used) on the RLSsignal is weak. When the NaCl concentration reached 1.5%,the difference of RLS intensity between morin-BSA andmorin decreased about 29%. The main reason is that theRLS intensity of morin increased about 25% (for 1.5%NaCl), which is due to the aggregation of morin by addi-tion of salts [33]. Most metal ions also do not interferethis assay. But 4 µmol/L Al3+ enhances the RLS signaldoubly, this may be explained by the formation of aternary Al3+ – morin – BSA complex at pH 4–5 and thisresulted in a great enhancement of RLS. EDTA, oxalicacid, ethanol also significantly affect the RLS signal. Theinfluence of ethanol on RLS is shown in Fig.4. When theconcentration of ethanol is more than 12.5% (v/v), the in-tensity of RLS decreases steeply. This result agrees withthe Rayleigh formula. Because nethanol, 20°C = 1.36048, nwater, 20°C = 1.33299 [34], addition of ethanol will increasethe refractive index of the medium, and thus the RLS sig-nal became smaller. Cu2+, Cr3+ also enhance the signal,however, Al3+, Cu2+, Cr3+ did not interfere in the real assayof protein when their concentration are lowered to 0.04 µmol/L (still higher than the concentration in humanplasma).

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Fig.2 Effect of pH on the RLS intensity of a mixture of morin-BSA (M). Morin: 0.009%, BSA: 3.58 µg/mL

Fig.3 Effect of surfactants on the RLS intensity. CTAB: morin(p) and morin-BSA (P); SDS: morin (g) and morin-BSA (G);Triton X-100: morin (m) and morin-BSA (M). Morin: 0.009%;BSA: 1.97 µg/mL

Standard regression equation for proteins

The application of the method for several proteins, suchas BSA, HSA, γ-G, chymotrypsin, cytochrome, lysozyme,pepsin, protamine sulfate, hemoglobin, egg albumin,

could be seen from Fig.5. The standard regression equa-tions for variant proteins are shown in Table 2. Differentproteins have different isoelectric points. At the sametime, the weight, size and shape of the molecules are alsodifferent, so the RLS signals for various proteins are dif-ferent.

Sample determination

BSA is similar to HSA in structure and biological func-tion, and HSA is more expensive than BSA, thereforeBSA was used to optimize the conditions for the determi-

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Table 1 Effect of interfering substances

Substance Concentration Change of RLS intensity(%)

L-Tyrosine 20 µg/mL 9.0L-Tryptophan 20 µg/mL –4.3L-Lysine 20 µg/mL –4.8L-Asparagine 20 µg/mL –0.4L-Phenylalanine 20 µg/mL –4.3L-Serine 20 µg/mL 1.0L-Glutamine 20 µg/mL –1.7L-Leucine 20 µg/mL 6.1L-Histidine 20 µg/mL 6.9L-Arginine 20 µg/mL 0L-Cysteine 20 µg/mL 2.6L-Proline 20 µg/mL 3.0Mg2+(nitrate) 4 µmol/L –7.4Fe3+(nitrate) 4 µmol/L 6.0Cd2+(nitrate) 4 µmol/L –5.2Al3+(sulfate) 4 µmol/L 93

0.04 µmol/L 21Mn2+(sulfate) 4 µmol/L 4.0Pb2+(nitrate) 4 µmol/L 10.0Cr3+(chloride) 4 µmol/L 18.0

0.04 µmol/L –0.4Cu2+(nitrate) 4 µmol/L 30

0.04 µmol/L –1.7Zn2+(chloride) 4 µmol/L 4.0Ca2+(nitrate) 4 µmol/L 1.0KCl 5 mmol/L 6.0Ethanol 50% (v/v) –66.2Oxalic acid 10 mmol/L –19.9NH4Cl 50 mmol/L 9.0NH4NO3 50 mmol/L 4.0EDTA 2 mmol/L 11.2Urea 0.2 mol/L 3.0

BSA: 3.97 µg/mL

Table 2 Standard regression equation of proteins

Protein Standard regression r Linear equation range(C µg/mL) (µg/mL)

Protamine sulfate y = 15.32 + 10.14 C 0.9902 0.50– 3.01Hemoglobin y = 13.11 + 3.272 C 0.9904 1.7 – 8.45Pepsin y = 8.008 + 1.590 C 0.9988 0.72– 3.62BSA y = 9.056 + 3.885 C 0.9972 0.45– 7.15HSA y = 10.23 + 2.728 C 0.9982 0.46–11.14γ-G y = 12.12 + 2.503 C 0.9925 0.92– 7.33Cytochrome y = 11.83 + 9.520 C 0.9987 0.81– 6.50

Table 3 Comparison of the morin assay and the CBB assay forprotein in human plasma a (from adults)

Serum sample Protein (mg/mL, found in human plasma)

Morin assay CBB assay

1 67.2 62.02 71.8 66.03 63.5 57.24 98.3 101.75 63.8 65.2

a Obtained from the Hospital of Peking University

Fig.4 Effect of ethanol on the RLS intensity of morin-BSA (M).Morin: 0.009%; BSA: 1.97 µg/mL

Fig.5 RLS response pattern for various proteins. BSA (p), HSA(G), γ-G (h), Chymotrypsin (+), Cytochrome (×), Protamine sul-fate (M), Hemoglobin (m), Lysozyme (P), Pepsin (r), Egg albu-min (R). Morin: 0.009%

nation, and HSA was used for the standard curve. Fivesamples were assayed according to the general procedure,and the results were compared with Commassie brilliantblue (CBB) method (see Table 3). The results obtainedwith the morin method are close to those obtained withthe CBB method, so it can be clearly shown that this newmethod could be used for the analysis of real samples.

Conclusion

The mechanism of the enhancement of RLS by proteins isstill under study, but that this novel spectrophotometrycould be used in protein determination is undoubted. Theresults compare well with the Acid Chrome Blue K method[15], the Bromo-Pyrogallol Red method [16] and the Bro-mophenol Blue method [17]. The main advantage of theenhanced RLS technique is its high sensitivity and its sim-plicity in handling in an ordinary biochemical laboratory.In this paper, the reaction of morin with BSA by electro-static forces is described. The addition of BSA to morinhardly changes the absorption spectrum both in intensityand wavelength, but the RLS intensity of morin was en-hanced significantly. The reason for this may be an ex-tended array of electronically coupled chromophores hav-ing a large oscillator strength, which enhances the RLS,but the sensitivity of the UV-VIS spectrum is too low toobserve such microenvironmental changes due to elec-tronically coupling [13]. Therefore, a new protein spectralprobe was developed. The same principle could be the ba-sis for other new spectral probes for proteins.

The main proteins in human serum are HSA and γ-G,and both proteins give response to the morin method andthe CBB method, so this assay is comparable with theCBB method in the protein determination of humanserum. The linear range of BSA and HSA is 0.45–7.15and 0.46–11.14 µg/mL, respectively. The sensitivity ofthe morin method for protein determination is obviouslyhigher than that of most spectrophotometric methods,such as the Lowry method, which can be used only forprotein concentrations of at least 5 µg/mL [1]. Though theCBB method is the commonly used assay for protein de-termination, the adherence of the Commassie brilliantblue reagent at the glass is difficult to overcome. There-fore, the washing of the tubes and of the cuvette is labori-ous. The morin method avoids this drawback.

Acknowledgment This work was supported by the National Nat-ural Science Foundation of China (NSFC) and Doctoral ProgramFoundation of Higher Education of China, all the authors here ex-press their deep thanks.

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