Cuprolinic Blue: a specific dye for single-stranded RNA in the presence of magnesium chloride. I. Fundamental aspects

Summary Qualitative and quantitative aspects of the cationic dye Cuprolinic Blue were investigated with model films of polyacrylamide gel in which RNA, DNA and other biological polyanionic compounds had been incorporated. In the presence of 1m MgCl₂, Curpolinic Blue was found to bind specifically to single-stranded RNA, leaving native DNA, proteins, (acid) polysaccharides and phospholipids completely unstained. Under these conditions, Cuprolinic Blue is complexed by non-electrostatic bonds with non-stacked purine bases, mainly adenine. Optimal conditions for dye binding and differentiation have been defined. Both the Cuprolinic Blue-MgCl₂ staining of single-stranded RNA and the Cuprolinic Blue staining of RNA and DNA in the absence of MgCl₂ were found to obey the Lambert-Beer law. The advantages and possible applications of Cuprolinic Blue are compared with well-known (indirect) histochemical RNA staining procedures.     

Cuprolinic Blue: a specific dye for single-stranded RNA in the presence of magnesium chloride. II. Practical applications for light microscopy

The application of the new nucleic acid dye Cuprolinic Blue to cell smears and tissue sections has been described. Without added cations, Cuprolinic Blue stains both DNA and RNA, whereas in the presence of 1 M MgCl2, Cuprolinic Blue specifically stains single-stranded RNA only. The total RNA can be stained after removal of DNA by DNAase digestion. Fixation in a modified Carnoy solution gave optimal staining results in all cases tested. By cytophotometry, a reliable and reproducible relative estimate can be obtained of the total nucleic acid content, the total RNA content and the amount of single-stranded RNA alone per cell.     

Cleavage of DNA by electrochemically activated MnIII and FeIII complexes of meso-tetrakis (N-methyl-4-pyridiniumyl)porphine

Electrochemical methods were used to activate MnIII and FeIII complexes of meso-tetrakis(N-methyl-4-pyridiniumyl)porphine (H2TMPyP) to cause cleavage of pBR322 DNA and to study their interaction with sonicated calf thymus DNA. Electrochemical reduction of MnIIITMPyP and FeIIITMPyP (at low concentrations) in the presence of O2 was required to activate these complexes. However, FeIIITMPyP at 1 x 10(-6) M produced DNA strand breakage without being electrochemically reduced. At low concentrations, FeIITMPyP was more efficient at cleaving DNA than MnIITMPyP. Reduction of O2 at a platinum electrode also produced some cleavage but to a much smaller extent. The oxidized form of MnIIITMPyP (charge 5+) has higher affinity for sonicated calf thymus (CT) DNA than the reduced form (charge 4+), as determined by the negative shift in E degrees' for the voltammetric wave in the presence of DNA. Both forms of FeIIITMPyP (charge 4+) interact with DNA to about the same extent. Differential pulse voltammetry was used to determine binding constants (K) and binding-site sizes (s) of the interaction of these metalloporphyrins with sonicated CT DNA. The data were analyzed assuming both mobile and static equilibria. MnIIITMPyP binds to DNA (5 mM Tris, 50 mM NaCl, pH 7) with K = 5 (+/- 2) x 10(6) M-1, s = 3 bp (mobile) or K = 3.6 (+/- 0.3) x 10(6) M-1, s = 4 bp (static). FeIIITMPyP at that ionic strength caused DNA precipitation. At higher ionic strength (0.1 M Tris, 0.1 M NaCl, pH 7), FeIIITMPyP associates to DNA with K = 4.4 (+/- 0.2) x 10(4) M-1, s = 5 bp (mobile) or K = 1.9 (+/- 0.1) x 10(4) M-1, s = 6 bp (static).     

The binding mode of a mammalian (boar) protamine to DNA

The binding modes of mammalian and fish protamines to DNA were studied by reconstitution experiments from dansylated protamines and DNA, using fluorescence spectroscopy, thermal denaturation and sedimentation. Both boar and fish protamines showed strong positive cooperativity in binding to DNA. Binding parameters of the protamines were determined in 0.1 M NaCl, 50 mM Tricine-HCl, pH 7.4, at 37 degrees C: in the boar protamine, the cooperative binding constant (Kc) = 3.4 X 10(6) M-1 and the cooperative factor (q) = 667, in the fish protamine, Kc = 1.8 X 10(7) M-1 and q = 304. The boar protamines bound to DNA with two functional domains, but the fish protamines bound directly to DNA as a single linear molecule.     

Neither delta- nor lambda-tris(phenanthroline)ruthenium(II) binds to DNA by classical intercalation.

Equilibrium binding studies and viscosity experiments are described that characterize the interaction of delta- and lambda-[Ru(o-phen)3]2+ with calf thymus DNA. The mode of binding of these compounds to DNA is a matter of controversy. Both isomers of [Ru(o-phen)3]2+ were found to bind but weakly to DNA, with binding constants of 4.9 (+/- 0.3) x 10(4) M-1 and 2.8 (+/- 0.2) x 10(4) M-1 determined for the delta and lambda isomers, respectively, at 20 degrees C in a solution containing 5 mM Tris-HCl (pH 7.1) and 10 mM NaCl. We determined that the quantity delta log K/delta log [Na+] equals 1.37 and 1.24 for the delta and lambda isomers, respectively. Application of polyelectrolyte theory allows us to use these values to show quantitatively that both the delta and lambda isomers are essentially electrostatically bound to DNA. Viscosity experiments show that binding the lambda isomer does not alter the relative viscosity of DNA to any appreciable extent, while binding of the delta isomer decreases the relative viscosity of DNA. From these viscosity results, we conclude that neither isomer of [Ru(o-phen)3]2+ binds to DNA by classical intercalation.     

Interactions of pyronin Y(G) with nucleic acids

Spectral properties of pyronin Y(PY) alone or in complexes with natural and synthetic nucleic acids of various base compositions have been studied in aqueous solution containing 10 or 150 mM NaCl and 5 mM Hepes at pH 7.0. The dimerization constant (KD = 6.27 X 10(3), M-1) and the absorption spectra of the dye in monomeric and dimeric form were established. The complexes of PY with single-stranded (ss) nucleic acids show a hypsochromic shift in absorption, and their fluorescence is quenched by over 90% compared to free dye. In contrast, complexes with double-stranded (ds) RNA or DNA (binding by intercalation) exhibit a bathochromic shift in their absorption (excitation) spectrum, and their fluorescence is correlated with the base composition of the binding site. Namely, guanine quenches fluorescence of PY by up to 90%, whereas A, C, I, T, and U bases exert a rather minor effect on the fluorescence quantum yield of the dye. The intrinsic association constant of the dye to ds RNA (Ki = 6.96 X 10(4), M-1) and to ds DNA (Ki = 1.74 X 10(4), M-1) was measured in 150 mM NaCl; the binding site size was 2-3 base pair for both polymers. Implications of these findings for qualitative and quantitative cytochemistry of nucleic acids are discussed.     

Study of binding of some amino acid derivatives of 2,2'-bipyridineplatinum(II) to calf thymus DNA

The interactions of glycine, alanine, valine, leucine, proline, methionine, and asparagine derivatives of 2,2'-bipyridineplatinum(II) with calf thymus DNA, polyadenylic acid, and polyguanylic acid have been studied by difference absorption spectral technique. The association constants (Kapp) and number of binding sites per 100 nucleotides (n) have been obtained from binding isotherms which were constructed by treatment of data according to Scatchard equation. The Kapp values (2.2 X 10(4) to 4.6 X 10(5) M-1) of binding of platinum complexes with DNA are comparable to Kapp values (5.6 X 10(4) to 2.9 X 10(5) M-1) of binding of platinum complexes with polyguanylic acid. However, the polyadenylic acid does not exhibit any binding to platinum complexes. The hydrogen bonding is, however, the most probable mode of bonding involved in stabilizing the DNA-amino acid complexes.     

Cleavage of DNA with methidiumpropyl-EDTA-iron(II): reaction conditions and product analyses

The synthesis of methidiumpropyl-EDTA (MPE) is described. The binding affinities of MPE, MPE.Ni(II), and MPE.Mg(II) to calf thymus DNA are 2.4 X 10(4) M-1, 1.5 X 10(5) M-1, and 1.2 X 10(5) M-1, respectively, in 50 mM NaCl, pH 7.4. The binding site size is two base pairs. MPE.Mg(II) unwinds PM2 DNA 11 +/- 3 degrees per bound molecule. MPE.Fe(II) in the presence of O2 efficiently cleaves DNA and with low sequence specificity. Reducing agents significantly enhance the efficiency of the cleavage reaction in the order sodium ascorbate greater than dithiothreitol greater than NADPH. At concentrations of 0.1-0.01 microM in MPE.Fe(II) and 10 microM in DNA base pairs, optimum ascorbate and dithiothreitol concentrations for DNA cleavage are 1-5 mM. Efficient cleavage of DNA (10 microM in base pairs) with MPE.Fe(II) (0.1-0.01 microM) occurs over a pH range of 7-10 with the optimum at 7.4 (Tris-HCl buffer). The optimum cleavage time is 3.5 h (22 degrees C). DNA cleavage is efficient in a Na+ ion concentration range of 5 mM to 1 M, with the optimum at 5 mM NaCl. The number of single-strand scissions on supercoiled DNA per MPE.Fe(II) under optimum conditions is 1.4. Metals such as Co(II), Mg(II), Ni(II), and Zn(II) inhibit strand scission by MPE. The released products from DNA cleavage by MPE.Fe(II) are the four nucleotide bases. The DNA termini at the cleavage site are 5'-phosphate and roughly equal proportions of 3'-phosphate and 3'-(phosphoglycolic acid). The products are consistent with the oxidative degradation of the deoxyribose ring of the DNA backbone, most likely by hydroxy radical.     

Metachromatic staining and electron dense reaction of glycosaminoglycans by means of Cuprolinic Blue

Summary The cationic phthalocyanin-like dye Cuprolinic Blue, unlike phthalocyanin dyes such as Alcian Blue or Astra Blue, can definitely exhibit a clear metachromatic reaction with appropriate substrates, The application of Cuprolinic Blue to epoxy-embedded semithin sections revealed that mast cell cytoplasmic granules, goblet cell mucin and cartilage matrix stained in violet shades (metachromatic), whereas nuclear chromatin presented a bright blue coloration (orthochromatic). The metachromatic structures showed a high degree of contrast when ultrathin sections treated with Cuprolinic Blue were examined by electron microscopy.Cytophotometric measurements of stained components from the large intestine showed different absorption maxima: at 580 nm for mucin and at 640 nm for nuclei. The spectroscopical analysis revealed a clear-cut metachromatic shift when the dye was in the presence of chondroitin—4-sulphate. The addition of aluminium metal to Cuprolinic Blue solutions resulted in a striking spectral change; under such conditions the dye showed absorption maximum at 530 nm.     

Binding of Cu(II) to non-prosthetic sites in ceruloplasmin and bovine serum albumin

The binding of Cu(II) to native human, porcine, bovine and ovine ceruloplasmin (Cp) and to bovine serum albumin (bSA) has been studied at pH 7.4, 30 mM barbital buffer. The results were analyzed for the strength and the number of binding sites using Scatchard plots. Evidence for additional copper binding sites in Cp and bSA was obtained suggesting a role for copper ion in the homeostatic regulation of Cu(II) and other metal ions in the serum. In the binding studies the Cp was freed of exogenous Cu(II) by passing it over a Chelex-100 column. Two flow rates were used, 4 ml/hr and 40 ml/hr, which removed Cu(II) of different affinities. Cp passed at the slower flow rate (Cp4) only contained the prosthetic copper atoms. Cp passed at the faster flow rate (Cp40) contained one additional copper atom with a Ka approximately 10(7) M-1. Another 2-6 Cu(II) ion could be added to the Cp40 with an average affinity of about Ka approximately 10(5) M-1. The Cu(II) ions found in Cp provide two distinguishable classes: (1) the prosthetic copper atoms and (2) the exogenous copper atoms that can be removed by Chelex-100. For bSA one copper atom was bound strongly with a Ka value approaching 10(12) - 10(13) M-1 and was not removed by Chelex-100 at any flow rate. A second copper atom was found with a Ka = 5.2 x 10(6) M-1 and was removed by Chelex-100 at 4 ml/hr. Three additional copper atoms were bound with a Ka = 1.6 x 10(5) M-1; they were readily removed by Chelex-100 at 40 ml/hr but were nondialysable.     

Interaction of SV40 T-antigen with homologous and heterologous DNA

Using the DNA filter binding assay, the effects of ionic strength and pH on SV40 T-antigen interaction with viral DNA were studied. The apparent association constants for T-antigen binding to SV40 DNA in Scatchard coordinates in the presence of 40 mM NaCl are equal to 0.67 . 10(6) M-1 (pH 6.0) and 0.86 x 10(7) M-1 (pH 7.4). These data indicate that the interaction between T-antigen and SV40 DNA is more specific at pH 7.4. The coincident values of association constants for T-antigen binding to viral and cellular DNAs (Ka = 0.9 x 10(7) M-1 for cellular DNA) at pH 7.4 and the absence of competition between the two DNA species upon binding with T-antigen suggest that viral and cellular DNAs possess similar sites for T-antigen binding. Denatured DNA competes with viral DNA only at pH 6.0, when the T-antigen--SV40 DNA interaction is less specific.     

Cascade blue derivatives: water soluble, reactive, blue emission dyes evaluated as fluorescent labels and tracers.

Fluorescent dyes based on the pyrenyloxytrisulfonic acid (Cascade Blue) structure were prepared and evaluated. The dyes contain functional groups that react with amines, thiols, acids, aldehydes, and ketones, forming covalently bonded, fluorescent derivatives of molecules with broad biological interest. Reactive groups in the Cascade Blue dyes include carboxylic acids and activated esters, amines, hydrazides, alcohols, photoaffinity reagents, acrylamides, and haloacetamides. The dyes exhibited absorption maxima at 374-378 nm and 399-403 nm, with extinction coefficients in the range of 1.9 x 10(4)-2.4 x 10(4) M-1cm-1 and 2.3 x 10(4)-3.0 x 10(4) M-1cm-1, respectively. Emission maxima ranged from 422-430 nm. The spectral properties of the fluorescent dyes are sufficiently different from fluorescein to permit simultaneous use of both dyes with minimum spectral interference. The Cascade Blue derivatives have narrower spectral bandwidths and smaller Stokes' shifts than other reactive dyes with similar spectral properties, do not show appreciable sensitivity to pH, have higher solubilities in aqueous solution, and have good to excellent quantum yields. Cascade Blue conjugates of a number of histochemically and biologically useful molecules were prepared, including dextrans, albumins, Fc receptor binding proteins, antibodies, lectins, membrane receptor binding proteins, and biotin binding proteins, as well as biological particles and bacteria. Cascade Blue conjugates of secondary and tertiary labels yielded specific fluorescence localization in the indirect immunofluorescent staining of human epithelial cell (HEp-2) nuclei.     

Action mechanism of Escherichia coli DNA photolyase. I. Formation of the enzyme-substrate complex

Escherichia coli DNA photolyase (photoreactivating enzyme) is a flavoprotein. The enzyme binds to DNA containing pyrimidine dimers in a light-independent step and, upon illumination with 300-600 nm radiation, catalyzes the photosensitized cleavage of the cyclobutane ring thus restoring the integrity of the DNA. We have studied the binding reaction using the techniques of nitrocellulose filter binding and flash photolysis. The enzyme binds to dimer-containing DNA with an association rate constant k1 estimated by two different methods to be 1.4 X 10(6) to 4.2 X 10(6) M-1 S-1. The dissociation of the enzyme from dimer-containing DNA displays biphasic kinetics; for the rapidly dissociating class of complexes k2 = 2-3 X 10(-2) S-1, while for the more slowly dissociating class k2 = 1.3 X 10(-3) to 6 X 10(-4) S-1. The equilibrium association constant KA, as determined by the nitrocellulose filter binding assay and the flash photolysis assay, was 4.7 X 10(7) to 6 X 10(7) M-1, in reasonable agreement with the values predicted from k1 and k2. From the dependence of the association constant on ionic strength we conclude that the enzyme contacts no more than two phosphodiester bonds upon binding; this strongly suggests that the pyrimidine dimer is the main structural determinant of specific photolyase-DNA interaction and that nonspecific ionic interactions do not contribute significantly to substrate binding.     

Oxidative degradation of cationic metalloporphyrins in the presence of nucleic acids: a way to binding constants?

Mn(III) and Fe(III) complexes of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (M-TMePyP) and related hybrid molecules ("metalloporphyrin-ellipticine") were activated by potassium monopersulfate in the presence of variable calf thymus (CT) DNA and NaCl concentrations. Monitored by visible spectroscopy (Soret band), fast degradation of the free metalloprophyrin was observed while the DNA-bound form appeared protected. This direct quantitation of free versus bound metalloporphyrin ratios allowed determination of binding constants: Mn- and Fe-TMePyP respectively bind to CT DNA (5 mM phosphate buffer, 0.1 M NaCl, pH 7) with K = 3 X 10(4) and 1.2 X 10(4) M-1. Mn-TMePyP showed a greater affinity for poly[d(A-T)] (K = 1.2 X 10(5) M-1) than for poly[d(G-C)] (K = 0.2 X 10(4) M-1). This method allowed us access to the intrinsic DNA affinity of the metalloporphyrin moiety of the hybrid molecules "metalloporphyrin-ellipticine".     

Noncovalent DNA binding of bis(1,10-phenanthroline)copper(I) and related compounds

The noncovalent DNA binding of the bis(1,10-phenanthroline)copper(I) complex [(Phen)2CuI] was examined under anaerobic conditions by absorption and circular dichroism spectroscopy, and viscometry, as a function of phenanthroline concentration. Analyses according to the McGhee-von Hippel method indicated that binding exhibited both neighbor-exclusion and positive cooperativity effects, with a neighbor-exclusion parameter n approximately 2 and a cooperativity parameter omega approximately 4. The association constant for (Phen)2CuI binding decreased with increasing concentration of phenanthroline in excess over that required to stoichiometrically generate (Phen)2CuI, indicating that free phenanthroline was a weak competitive inhibitor of (Phen)2CuI binding. The maximal association constant for DNA binding of (Phen)2CuI in 0.2 M NaCl and 9.8% ethanol, extrapolated to zero concentration of excess phenanthroline, was 4.7 x 10(4) M-1 (DNA base pairs). The magnitude of the neighbor-exclusion parameter, the changes in spectral properties of (Phen)2CuI induced by DNA binding, and the increase in DNA solution viscosity upon (Phen)2CuI addition are consistent with a model for DNA binding by (Phen)2CuI involving partial intercalation of one phenanthroline ring of the complex between DNA base pairs in the minor groove as suggested previously [Veal & Rill (1989) Biochemistry 28, 3243-3250]. Viscosity measurements indicated that the mono(phenanthroline)copper(I) complex also binds to DNA by intercalation; however, no spectroscopic or viscometric evidence was found for DNA binding of free phenanthroline or the bis(2,9-dimethyl-1,10-phenanthroline)copper(I) complex. DNA binding of free phenanthroline may be cooperative and induced by prior binding of (Phen)2CuI.     

Staining of proteoglycans in mouse lung alveoli. I. Ultrastructural localization of anionic sites

Summary In order to contrast anionic sites, in mouse lung alveoli, two staining procedures were applied: (a) staining with Ruthenium Red and Alcian Blue and (b) staining with Cuprolinic Blue in a critical electrolyte concentration method. The Ruthenium Red-Alcian Blue staining procedure revealed electron-dense granules in the alveolar basement membrane. The granules were closely associated with the epithelial cell membrane and continued to stain even when the procedure was carried out at a low pH, indicating the presence of sulphate groups in the granules.After staining with Cuprolinic Blue, electron-dense filaments, also closely associated with the cell membrane, became visible in the basement membrane of type I epithelial cells. Their length depended on the MgCl₂ concentration used during staining. At 0.4m MgCl₂, the length was mostly within the range 100–180 nm. Using a modified Cuprolinic Blue method, the appearance of the filaments closely resembled that of spread proteoglycan monomers with their side-chains condensed. The basement membrane of type II epithelial cells also contained filaments positive towards Cuprolinic Blue; their length, however, was smaller in comparison with those of type I epithelial cells. The filaments lay in one plane and provided the whole alveolus with an almost continuous sheet of anionic sites. Cuprolinic Blue staining also revealed filaments in the basement membrane of the capillary endothelial cells. Furthermore, Cuprolinic Blue-positive filaments (average length about 40 nm) became apparent in close contact with collagen fibrils and separated from each other according to the main banding period of the collagen fibrils (about 60 nm), indicating a specific ultrastructural interaction between these two components. Filaments connecting collagen fibrils with each other were also detected.     

Drug-DNA interactions: spectroscopic and footprinting studies of site and sequence specificity of elliptinium.

The binding of the antitumoral ellipticine derivative 2-methyl-9-hydroxyellipticinium acetate (elliptinium; NMHE) to DNA was analyzed by the combined use of DNase I footprinting and spectroscopic methods. Using two fragments of pBR322 DNA, five discrete NMHE binding sites of 5-7 protected base pairs (bp) were detected by footprinting at 4 degrees C on the analyzed regions. These corresponded to alternating pyrimidines and purines. The inactive derivative 2-methyl ellipticinium acetate L(NME) lacking a hydroxy group failed to demonstrate DNA protection even at low temperature. Ultraviolet-absorption and 1H-nmr analysis was performed using two autocomplementary octanucleotides d(TGACGTCA) (I) and d(ACTGCAGT) (II). The uv-absorption titrations resulted in an intercalative binding mode for NMHE in the oligomers. Analysis of the derived biphasic Scatchard plots yielded two binding sites corresponding to approximately 6-bp and 2-bp sizes and characterized by apparent association constants K1 approximately 10(8) M-1 and K2 approximately 10(6) M-1, respectively. The 1H-nmr analysis of exchangeable (imino) protons and nonexchangeable protons performed in the one- and two-dimensional modes confirmed the intercalation of NMHE, and further revealed the existence of multiple sites on DNA. Assuming that imino resonance line width concerned the sole kinetic effects, 10-ms order lifetimes were estimated for the drug-oligonucleotide complexes at 7 degrees C, pH 7, and 0.1 ionic strength. Finally, examination of every drug-DNA spectra in the light of the footprinting results indicated that there was a preference for binding of NMHE to the CpG (octamer I) and TpG (octamers I and II) steps.     

Association of iron-protoporphyrin-IX (hemin) with myosins

Addition of myosins isolated from guinea pig heart and rabbit skeletal muscle to hemin solutions resulted in the appearance of new absorption spectra indicating association of hemin and the myosins. Binding stoichiometry based on absorption changes was found to be two hemin sites per myosin molecule. The binding constants calculated from quenching of the intrinsic fluorescence of the myosins by hemin are Ka = 7 (+/- 2) 10(6) M-1 for skeletal muscle myosin, and Ka = 3 (+/- 1) x 10(7) M-1 for heart muscle myosin. Based on these findings, myosins are suggested as potential transporters of free hemin between cell organelles.     

Binding of ellipticine base and ellipticinium cation to calf-thymus DNA. A thermodynamic and kinetic study

The acid-basic properties of ellipticine have been re-estimated. The apparent pK of protonation at 3 microM drug concentration is 7.4 +/- 0.1. The ellipticine free base (at pH 9, I = 25 mM) intercalates into calf-thymus DNA with an affinity constant of 3.3 +/- 0.2 X 10(5) M-1, and a number of binding sites per phosphate of 0.23. The ellipticinium cation (pH 5, I = 25 mM) binds also to DNA with a constant of 8.3 +/- 0.2 x 10(5) M-1 and at a number of binding sites (n = 0.19). It is postulated that the binding of the drug to DNA at pH 9 is driven by hydrophobic and/or dipolar effects. Even at pH 5, where ellipticine exists as a cation, it is thought that the hydrophobic interaction is the main contribution to binding. The neutral and cationic forms share common binding within DNA sites but yield to structurally different complexes. The free base has 0.04 additional specific binding sites per phosphate. As determined from temperature-jump experiments, the second-order rate constant of the binding of the free base (pH 9) is 3.4 x 10(7) M-1 s-1 and the residence time of the base within the DNA is 8 ms. The rate constant for the binding of the ellipticinium cation is 9.8 x 10(7) M-1 s-1 when it is assumed that drug attachment occurs via a pathway in which the formation of an intermediate ionic complex is not involved (competitive pathway).     

Binding of Streptomyces pepsin inhibitor (acetyl-pepstatin) with chymosin (Rennin)

Chymosin (Rennin) was effectively purified using an AH-Sepharose 4B column. Binding of Streptomyces pepsin inhibitor (acetul-pepstatin) with chymosin was studied spectroscopically. The binding caused ultraviolet difference and CD spectral changes suggesting microenvironmental changes around tryptophan and/or tyrosine residue(s) in chymosin. The fluorescence intensity of a hydrophobic probe, 2-p-toluidinylnaphthalene-6-sulfonate, increased in the presence of chymosin and was further amplified when Streptomyces pepsin inhibitor was added to the chymosin-2-p-toluidinylnaphthalene-6-sulfonate solution. The binding and dissociation-rate constants between chymosin and the inhibitor were determined using 2-p-toluidinylhnaphthalene-6-sulfonate as a probe. The binding constant was determined from the binding and dissociation-rate constants, to be 3.1 . 10(7) M-1 at 25 degrees C, pH 5.5.