Denaturation of glycinin by urea and guanidine hydrochloride

Denaturation of glycinin by urea and guanidine hydrochloride (GdnHC1) has been studied at 30° by viscosity and circular dichroism (CD) measurements. The kinetics of denaturation by 8M urea at 20, 30, 40 and 50°, and by 6M GdnHC1 at 20° have been measured by monitoring the increase in absorbance at 287 nm as a function of time. Viscosity increased with denaturant concentration and reached maximum value of 28 mL/g at 6M GdnHC1 and 7M urea. The (negative) molar ellipticity values in the region 250–200 nm decreased with increase in denaturant concentration. Analysis of viscosity and CD data indicated that both sets of data fitted the same curve of f_d (fraction of protein denatured) versus denaturant concentration. The kinetic data followed first order reaction kinetics, These suggest that denaturation of glycinin by urea/GdnHC1 is a two-state process and follows the same pattern as that of globular proteins.     

Anomalous effect of denaturants on sulfitolysis of protein disulfide bonds

The effect of three protein denaturants, urea, guanidinium hydrochloride and guanidinium thiocyanate on the cupric ion-catalyzed oxidative sulfitolysis of disulfide bonds of glycinin was studied by following the kinetics of the reaction. The rate constant for sulfitolysis increased with the concentration of each denaturant and reached constancy at 8M urea, 6M guanidinium hydrochloride and 6M guanidinium thiocyanate, but the extent of sulfitolysis at the final denaturant concentrations was different. The reaction rate followed the order 8M urea > 6M guanidinium hydrochloride > 6M guanidinium thiocyanate as compared to the control sulfitolysis reaction. Determination of thermodynamic activation parameters revealed a higher positive free energy of activation in the case of 6 M guanidinium salts compared to 8 M urea. This was contributed by a higher negative entropy of activation causing the reaction to proceed slowly. The increase in the negative entropy of activation could arise from the repulsion of SO⁼₃ ions from the more negatively charged surface of the protein in the presence of guanidinium salts compared to urea. This in turn might arise from the preferential binding of the anion components of the guanidinium salts.     

Interaction of promazine with bovine serum albumin and hexadecyltrimethylammonium bromide using light absorption and fluorometric techniques

The association of promazine with bovine serum albumin and ammonium detergent micelle was measured using the changes in the u.v. absorption and the fluorescence emission of the interactants. The endogenous fluorescent properties of promazine were not altered upon binding to bovine serum albumin. The association of promazine with protein could be determined by measuring the drug-induced fluorescence quenching of tryptophan emission. Such fluorescence-quenching reaction was pH sensitive. In bovine serum albumin binding of promazine, there was an increase in the extinction coefficient of the drug when associated with protein. Promazine binding to serum protein produced a hyperchromic change in the drug's absorption spectrum which paralleled the fluorescence-quenching reaction. The involvement of carboxyl groups in binding of promazine to protein molecules and the effect of the carboxyl groups on the emissions of both tyrosine and tryptophan are suggested. A slight increase in both absorption and fluorescence of promazine was observed when associated with detergent micelle. The addition of the detergent caused an increase in the extinction coefficient of promazine and a shift to longer wavelength of the absorption maximum.     

Risperidone interacts with serum albumin forming complex

The aim of the work is to study the mechanisms of the interaction of risperidone with human and bovine serum albumins using the fluorescence quenching technique. Risperidone is an atypical antipsychotic drug used to treat many psychiatric disorders. We selectively excited the fluorescence of tryptophan residues with a 290 nm wavelength light, and observed quenching by titrating human and bovine serum albumin solutions with risperidone. Emission spectra were recorded in the range from 300 to 450 nm for each quencher addition. Stern-Volmer graphs were plotted and quenching constants were estimated. Results showed that the drug quenches the fluorescence of the human serum albumin by the formation of a complex risperidone-albumin. Association constants calculated from Stern-Volmer equation for low concentrations (lower than 1:10 ratio risperidone/albumin) were of 2.56 × 10(5)M(-1), at 25 °C, and 1.43 × 10(5)M(-1), at 37 °C. As the quenching intensity of bovine serum albumin, which contains two tryptophan residues, was found to be higher than that of human serum albumin, which contains only one tryptophan residue. Hence, we suggest that the primary binding site for risperidone in albumin should be located in sub domain IB.     

DISSOCIATION AND DENATURATION BEHAVIOUR OF SESAME α-GLOBULIN IN SODIUM DODECYL SULPHATE SOLUTION*

The effect of anionic detergent, sodium dodecyl sulphate, on the major protein, α-globulin of sesame seed (Sesamum indicum L.) has been investigated by gel filtration, sedimentation velocity, viscosity, optical rotation, difference spectra and fluorescence measurements. The detergent causes dissociation of the protein first and then denaturation. In the detergent concentration range of 1.75–4.0 times 10^-3M four components are observed in the ultracentrifuge. The specific rotation of the protein increases with the detergent concentration above 2.5 times 10^-3M and shows a cooperative transition between 3–8 times 10^-3M detergent suggesting conformational change; above 8 times 10^-3M detergent the value of –[α] does not change. The reduced viscosity η_red however, increases above 2.5 times 10^-3M detergent and does not attain a plateau value. The difference spectrum of the protein indicates that both tryptophan and tyrosine groups have been affected by the detergent. The fluorescence intensity decreases and the maxima shifts towards red in the detergent solution resulting in an ‘isoemissive point’ at 355 nm. The double difference spectra in sucrose-detergent protein system show that below 5.0 times 10^-3M detergent, the difference absorption and fluorescence spectrum result from the binding of the detergent near the chromophoric groups and are not due to conformational change. Binding studies by equilibrium dialysis indicate the presence of 50 binding sites in the protein and a binding constant of 3.0 times 10³.     

Measurement of the Kinetics of Protein Unfolding in Viscous Systems and Implications for Protein Stability in Freeze-Drying

Purpose The aim of the study is to determine the degree of coupling between protein unfolding rate and system viscosity at low temperatures in systems relevant to freeze-drying.Methods The cold denaturation of both phosphoglycerate kinase (PGK) and β-lactoglobulin were chosen as models for the protein unfolding kinetics study. The system viscosity was enhanced by adding stabilizers (such as sucrose), and denaturant (guanidine hydrochloride or urea) was added to balance the stabilizing effect of sucrose to maintain the cold denaturation temperature roughly constant. The protein unfolding kinetics were studied by both temperature-controlled tryptophan emission fluorescence spectroscopy and isothermal high-sensitivity modulated differential scanning calorimetry (MDSC) (T_zero). Viscometers were used to determine the system viscosity. To verify the predictions of structure based on protein unfolding dynamics, protein formulations were freeze-dried above the glass transition temperatures, and the protein structures in dry products were determined by fluorescence spectroscopy of reconstituted solids by extrapolation of the solution data to the time of reconstitution.Results Empirical equations describing the effect of sucrose and denaturant (urea and guanidine hydrochloride) on protein cold denaturation were developed based on DSC observations [X. C. Tang and M. J. Pikal. The Effects of Stabilizers and Denaturants on the Cold Denaturation Temperature of Proteins and Implications for Freeze-Drying. Pharm. Res. Submitted (2004)]. It was found that protein cold denaturation temperature can be maintained constant in system of increasing sucrose concentration by simultaneous addition of denaturants (urea and guanidine hydrochloride) using the empirical equations as a guide. System viscosities were found to increase dramatically with increasing sucrose concentration and decreasing temperature. The rate constants of protein unfolding (or the half-life of unfolding) below the cold denaturation temperature were determined by fitting the time dependence of either fluorescence spectroscopy peak position shift or DSC heat capacity increase to a first-order reversible kinetic model. The half-life of unfolding did slow considerably as system viscosity increased. The half-life of PGK unfolding, which was only 3.5 min in dilute buffer solution at −10°C, was found to be about 200 min in 37% sucrose at the same temperature. Kinetics of protein unfolding are identical as measured by tryptophan fluorescence emission spectroscopy and by high-sensitivity modulated DSC. The coupling between protein unfolding kinetics and system viscosity for both proteins was significant with a stronger coupling with PGK than with β-lactoglobulin. The half-lives of PGK and β-lactoglobulin unfolding are estimated to be 5.5 × 10¹¹ and 2.2 years, respectively, even when they are freeze-dried in sucrose formulations 20°C above T_g′. Thus, freeze-drying below T_g′ should not be necessary to preserve the native conformation. In support of this conclusion, native PGK was obtained after the freeze-drying of PGK at a temperature more than 60°C above the system T_g′ in a thermodynamically unstable system during freeze-drying.Conclusions Protein unfolding kinetics is highly coupled with system viscosity in high viscosity systems, and the coupling coefficients are protein dependent. Protein unfolding is very slow on the time scale of freeze-drying, even when the system is freeze-dried well above T_g′. Thus, it is not always necessary to freeze-dry protein formulations at temperature below T_g′ to avoid protein unfolding. That is, protein formulations could be freeze-dried at product temperature far above the T_g′, thereby allowing much shorter freeze-drying cycle times, with dry cake structure being maintained by the simultaneous use of a bulking agent and a disaccharide stabilizer.     

Conformation and stability of the chymotrypsin inhibitor from winged bean seed (Psophocarpus tetragonolobus (L.) DC)

Spectrophotometric measurement was found to be a sensitive method for evaluating the stability of the chymotrypsin inhibitor from the winged bean. The thermal stability of this protein in aqueous solution was much greater at pH 3 than at pH 8 or pH 11. Evidence from u.v. absorption and from circular dichroism indicated that irreversible conformation changes occurred at higher temperature (> 70°). Circular dichroism and optical rotatory dispersion studies at pH 8 show that the inhibitor is rich in β-structure and virtually devoid of α-helix in aqueous solution. We conclude from experiments with denaturing solvents that the inhibitor is very stable and that high concentrations of denaturant are required before unfolding occurs. Chemical modification experiments with tetranitromethane were consistent with a tight stable structure; even in 6 M guanidine hydrochloride only three of the five tyrosine residues in the inhibitor molecule were nitrated. However, tyrosine does not seem to be implicated at the reactive site of the inhibitor. Interaction of the inhibitor with α-chymotrypsin and chymotrypsin B was also followed by difference spectroscopy in the ultraviolet region. Difference spectra were detected that were characteristic of changes in the environment of both tyrosine and tryptophan chromophores. Comparison of the spectral data obtained for the interaction of the inhibitor with bovine α-chymotrypsin and with chymotrypsin B indicated that a tryptophan residue may be involved at the reactive site of the inhibitor. Spectral changes were also detected for the interaction between the chymotrypsin inhibitor and trypsin, although it is well established that the specificity of this inhibitor is restricted to the chymotrypsins. However the complex formed between the inhibitor and the chymotrypsins (verified to have a 1:2 inhibitor: enzyme stoichiometry) was found to be stable to at least 50°, whereas the weaker “non-specific” interaction observed between the inhibitor and trypsin was largely destroyed on heating to 40°.     

FLUORESCENCE SPECTRA OF LYSOZYME EXCITED AT 305 NM IN PRESENCE OF UREA

Tryptophan 108 of hen egg white lysozyme was selectively excited at 305 nm and fluorescence spectra were recorded as a function of pH (2–9) and concentration of urea (0–8 M). Urea at low concentrations (1–4 M) quenches markedly the Trp 108 fluorescence around pH 7; the Λ_max, however, remains unaltered. The fluorescence quenching by urea is most likely due to local conformational changes around Trp 108 in the active site region of the enzyme. Substantial unfolding of the enzyme, however, was brought about by 4M urea below pH 3, and by 7 M urea at pH 10.3, as indicated by a marked red shift in the Λ_max of the fluorescence emission.     

荧光光谱法研究过氧化氢酶溶液稳定性

目的考察不同环境因素对过氧化氢酶(CAT)活性和构象的影响,为蛋白质制剂过程提供依据。方法改变温度、pH、变性剂浓度,用紫外分光光度法考察CAT溶液活性变化,并用荧光光谱法研究其构象变化。结果CAT溶液25℃p、H 7时,活性及荧光强度都达到最大;温度升高,荧光强度逐渐下降;pH降低或升高,荧光最大发射波长均红移;低浓度变性剂对CAT活性中心有稳定作用;浓度增加,荧光谱带红移,活性降低甚至丧失;脲和盐酸胍对CAT荧光有动态猝灭作用。结论用荧光光谱法研究CAT溶液稳定性,方法简便、准确、可靠。     

SPECTRAL STUDIES ON TWO GENETIC FORMS OF THE HUMAN SERUM PROTEINASE INHIBITOR,ALPHA-1-ANTITRYPSIN*

Alpha-1-antitrypsin, the major inhibitor of proteolytic enzymes in human serum, was isolated from normal individuals (protease inhibitor type MM) and from those with an inherited deficiency (protease inhibitor type ZZ) of circulatory protein. The two proteins were compared by circular dichroism spectroscopy, and by fluorescence quenching experiments using anionic (I^-), and neutral (acrylamide) probes. Both proteins share a similar secondary structure, i.e. ?45–50%α-helix and 15–20%β-structure. Evidence was accumulated to show that the micro-environment in the vicinity of the three tryptophanyl residues is altered in Z form as compared to the M form as shown by (a) the absence of the positive dichroic band in the region 290–300nm of the circular dichroism spectra, (b) a > 50% increase in quantum yield in the tryptophanyl fluorescence emission spectra, (c) an increased accessibility of tryptophan to quenching by iodide, and (d) acrylamide quenching experiments which indicate that all tryptophanyl residues in the Z protein are quenched equally or that quenching is dominated by a single residue, while in the M protein, heterogeneous quenching occurs. The potential significance of these findings in terms of alpha-1-antitrypsin deficiency state are discussed.     

REACTION OF BOVINE AND EQUINE GROWTH HORMONES WITH TETRANITROMETHANE

Bovine and equine growth hormones were chemically modified with tetranitromethane, at pH 7.4 during 5 h and at pH 8.0 in the presence of 8 M urea during 1 h. a) Both hormones have very similar but not identical reactivities. b) The nitration of the reactive tyrosines and tryptophan residues at pH 7.4 produces no detectable changes in their immunological or somatotrophic activities. c) The nitration of all tyrosine residues in both hormones gives rise to a complete loss of somatographic activity with no alteration of the immunological activity.     

FLUORESCENCE PROPERTIES OF NEUTRAL PROTEASE FROM BACILLUS SUBTILIS

The fluorescence properties of neutral protease from B. subtilis have been investigated under a variety of conditions and the results compared with those previously reported for the homologous metalloendopeptidase thermolysin (Fontana et al., 1977). In the pH range 5–9 neutral protease displayed a quite unusual fluorescence emission spectrum with a maximum near 320 nm, when excitation was at 295 nm. At this wavelength the protein fluorescence is due to tryptophan residues only, which, considering their blue-shifted emission, appear rather buried in the hydrophobic protein interior. Specific removal of the functional zinc ion from the enzyme with tetraethylenepentamine does not lead to alteration of the microenvironment around tryptophan residues. On the other hand, removal of both zinc and calcium with ethylenediaminetetraacetic acid brings these residues in full contact with the aqueous solvent medium. Fluorescence quenching measurements were also used to determine the exposure of tryptophan residues in the native enzyme as well as in the presence of chelating agents and protein denaturants. Melting profile experiments carried out by monitoring the fluorescence intensity at 320 nm indicated a cooperative transition at 60–70°. Temperature effects were also determined under conditions perturbed with respect to pH, guanidine hydrochloride and chelating agents. The results reveal differences in the fluorescence properties of the tryptophanyl residues of B. subtilis neutral protease relative to those of thermolysin, which are interpretable considering the location of these residues in the sequences of the two homologous proteins.     

A novel rhodamine-riboflavin conjugate probe exhibits distinct fluorescence resonance energy transfer that enables riboflavin trafficking and subcellular localization studies

Riboflavin (vitamin B2, RF) is taken up in eukaryotic cells via specialized transport mechanisms. Although RF has fluorescence properties, direct microscopic visualization of RF uptake and trafficking has been complicated by cellular autofluorescence. We describe the synthesis, cellular uptake characteristics, and spectroscopic properties of a novel rhodamine-riboflavin conjugate (RD-RF), including absorption and emission spectra, two-photon excitation spectra, and fluorescence pH dependence. The conjugate has a molar extinction coefficient of 23 670 M(-1) cm(-1) at 545 nm (excitation wavelength) with a fluorescence quantum yield of 0.94. This compound exhibits intramolecular fluorescence resonance energy transfer (FRET). Selective quenching of the FRET signal is observed when RD-RF is bound with high affinity by the chicken riboflavin carrier protein. In addition to the typical rhodamine excitation and emission, FRET provides a secondary signal for conjugate localization and an in situ mechanism for observing riboflavin binding. Solution and in vitro stability determinations indicate that the linkage between riboflavin and rhodamine is stable for the duration of typical pulse--chase and cellular trafficking experiments. The distinct spectroscopic properties of RD-RF together with a comparable affinity for RF-binding proteins render it an excellent tool for the study of RF transport and trafficking in living cells.     

Interaction in the cerebral metabolism of the biogenic amines: Effect of intravenous infusion of L-tryptophan on tryptophan and tyrosine in brain and body fluids

1. The distribution of the amino-acids tryptophan and tyrosine has been determined in plasma ultrafiltrate, whole plasma, erythrocytes, cerebrospinal fluid (c.s.f.) and various regions of the brain in dogs.2. The effect of tryptophan administration on the distribution of both these amino-acids showed that the alterations produced in tryptophan concentration did not appear to change the concentrations of tyrosine from their normal pattern.3. The implications of these results with regard to amino-acid transport systems in man and dog are discussed.     

Effect of pH and light on aggregation and conformation of an IgG1 mAb

During the purification process, monoclonal antibodies may be exposed to parts of UV-C (200 to 290 nm), UV-B (290 to 320 nm) and visible light (400 to 760 nm) under a variety of buffer and pH conditions. Together, these conditions can promote both chemical and physical degradation which may result in conformational changes. To examine this possibility, an IgG1 mAb at pH 3.5, 5, and 8 was exposed to UV light at multiple protein concentrations. Exposure to 302 nm light resulted in a pH-dependent formation of high molecular weight species where the degree of oligomerization increased with increasing pH. Characterization by SDS-PAGE under reducing and nonreducing conditions and size exclusion MALS revealed that the predominant species were nonreducible dimeric, trimeric and higher order oligomeric species which occurred through processes other than intermolecular disulfide bond formation. Biophysical characterization by differential scanning calorimetry demonstrated an overall loss of heat capacity suggesting a loss of conformational integrity with light exposure. A decrease in tryptophan fluorescence was paralleled by a significant decrease in the transition temperature measured during heat-induced unfolding following light exposure, also suggesting a significant change in conformational integrity. The observations by fluorescence spectroscopy coincided with pH-dependent changes in the alterations of secondary structure characterized by Fourier transform infrared spectroscopy and far-UV circular dichroism with the most acidic pH showing the greatest degree of change in the β-sheet structure. Exposure to UV light resulted in aggregation with pH-dependent yields decreasing in the order 8.0 > 5.0 > 3.5, while the opposite trend was observed for conformational changes, with pH-dependent extents decreasing in the following order 3.5 > 5.0 > 8.0. These pH-dependent trends suggest that different strategies will be required to stabilize the protein against these modifications during processing.     

Absorption and fluorescence study of the interaction between (2-hydroxy-benzimido)ethyl-n-hexylselenide and bovine serum albumin

The binding of Schiff base selenide, (2-hydroxy-benzimido)ethyl-n-hexylselenide, to bovine serum albumin (BSA) was studied using fluorescence spectroscopy. The measurement was performed in Tris-HCl buffer aqueous medium at pH 7.4. Stern-Volmer graphs were plotted and quenching constants were estimated. The quenching constant at 303 K was (1.639 +/- 0.046) x 10(13) L mol(-1) s(-1). Decreased quenching was observed as temperature increased, but at the temperature range of 303-313 K, the association of Schiff base selenide to BSA was not significantly different. The static quenching presented in the system of Schiff base selenide and BSA. A complex was possibly formed between Schiff base selenide and BSA, which was responsible for the quenching of the fluorescence of BSA. This fact was also confirmed by differences in the absorption spectra of BSA before and after Schiff base selenide addition. The hydrophobic interaction was found to play a main role in the binding according to the thermodynamic parameters, enthalpy change (DeltaH) and entropy change (DeltaS) of reaction. Schiff base selenide most likely binds to the hydrophobic pockets within sub domain IIA of BSA, which can be proved by competition experiments for sodium dodecyl sulfate. By constant-wavelength synchronous fluorescence spectra, the influence of (2-hydroxy-benzimido)ethyl-n-hexylselenide on the surrounding environment of tyrosine and tryptophan residues in BSA was also investigated. The red shift of the fluorescence peak of tryptophan residues indicated that the hydrophobic amino acid structure surrounding tryptophan residues in BSA collapsed slightly after the addition of (2-hydroxy-benzimido)ethyl-n-hexylselenide.     

UREA AND GUANIDINE HYDROCHLORIDE INDUCED DISSOCIATION AND DENATURATION OF BACTERIOPHAGE F2 CAPSID

A single, low molecular weight protein is found after urea or guanidine hydrochloride (Gdn.HCl) treatment of empty capsids derived from bacteriophage f2. The final product of denaturation is apparently a monomer, existing as a random coil in ≥ 4.0 M Gdn.HCl but in a less extended form in 8.0 M urea. In contrast, an 11 S protein component is isolated after treatment of the intact virus with 4.0 M Gdn.HCl (Zelazo & Haschemeyer, 1969), indicating that RNA plays a role in stabilizing larger subunits. Denaturation by Gdn.HCl occurs in two stages as measured by changes in CD and Stokes radius: dissociation that involves a structural perturbation of aromatic side chains, followed by a major, cooperative transition that evidently results in the loss of all noncovalent structure. Denaturation by urea appears to be a much less cooperative process that occurs in several steps over a wide range of urea concentration (1–7 M). In both urea and Gdn.HCl, dissociation into subunits begins at a lower concentration of denaturant than the major changes in conformation.     

Inhibition of rat brain tryptophan hydroxylation with p-chloroamphetamine.

p-Chloroamphetamine (PCA) at high concentrations (0.0025 to 0.02 M) inhibited rat brainstem tryptophan hydroxylase and, to a lesser extent, rat corpus striatum tyrosine hydroxylase. Hog kidney aromatic l-amino acid decarboxylase was not affected. Inhibition of tryptophan hydroxylase by p-chloroamphetamine was competitive with tryptophan and noncompetitive with 6,7-dimethyl-5,6,7,8-tetrahydropterine (DMPH₄). In rats given p-chloroamphetamine, brainstem tryptophan-hydroxylating activity was only slightly reduced, whereas striatal tyrosine-hydroxylating activity was not altered. Synthesis rates of brain serotonin and dopamine in rats treated with p-chloroamphetamine, estimated by measuring the accumulation of 5-hydroxytryptophan and dopa, respectively, after blockade of decarboxylase, were both decreased. In view of the high concentrations of p-chloroamphetamine required to inhibit tryptophan and tyrosine hydroxylases in vitro, mechanisms other than inhibition of enzyme by PCA might be responsible for the decelerated synthesis of serotonin and dopamine in brain (e.g. blockade of monoamine re-uptake).     

Application of Second-Derivative Fluorescence Spectroscopy to Monitor Subtle Changes in a Monoclonal Antibody Structure

In this study, the tertiary structure of a monoclonal antibody was analyzed under thermal and chemical stresses using second‐derivative fluorescence spectroscopy. The effect of polyols, sucrose, and ethylene glycol on the tertiary structure of monoclonal antibody‐U (mAb‐U) (pH 7.0) was studied under thermal stress (25°C–75°C). The tertiary structure of mAb‐U was also analyzed upon chemical denaturation using urea (2.0–8.0 M). The second derivative of mAb‐U showed three bands corresponding to the three spectral classes of tryptophan, class I (330 nm), class II (340 nm), and class III (350 nm). Class II was higher in intensity in the presence of polyols compared with the solution without any polyol. Thermally denatured structure of mAb‐U in sucrose and ethylene glycol was distinctly different than that in buffer. Addition of urea resulted in a decrease in intensity of class I and II, and an increase in intensity of class III implying unfolding. This study showed that second‐derivative fluorescence spectroscopy is an effective tool to monitor subtle alterations in the tertiary structure of proteins. The unfolding of a protein is reflected as an increase in the intensity of the polar class III accompanied with a decrease in the intensity of class I.     

HPLC法同时测定人血清中犬尿氨酸和色氨酸的浓度

目的:建立同时测定人血清中的犬尿氨酸和色氨酸浓度的方法。方法:血清样品用24%高氯酸以10:1进行蛋白沉淀处理,取上清液进样分析。采用高效液相色谱-紫外/荧光检测器法分别检测犬尿氨酸和色氨酸浓度,色谱柱为Accurasil-C18,流动相为乙酸钠-乙腈溶液(24.8mmol.L-1乙酸钠,用乙酸调pH值至约4.0)=90:10,流速1.0mL.min-1,紫外检测波长为360nm,荧光检测激发波长为285nm、发射波长为365nm。结果:犬尿氨酸、色氨酸血药浓度分别在0.16～20.89(r=0.9999)、0.24～61.58μmol.L-1(r=0.9996)范围线性关系良好;高、中、低3种浓度的方法回收率分别为91.33%～101.82%、91.49%～104.37%;日内及日间RSD均<6%。结论:本方法稳定、可行,可为临床疾病的诊断和药物治疗提供实验室依据。