Isolation and partial characterization of the N-terminal functional unit of subunit RtH1 from Rapana thomasiana grosse hemocyanin

Two different structural subunits were identified in Rapana thomasiana hemocyanin: RtH1 and RtH2. RtH1-a is the N-terminal functional unit in the subunit RtH1 and its stability toward temperature and chemical denaturation by guanidinium hydrochloride (Gdn.HCl) are studied and compared with the structural subunit RtH1 and the whole Rapana hemocyanin molecule. The conformational changes, induced by the various treatments, were monitored by CD and fluorescence spectroscopy. The critical temperatures (T(c)) for RtH1-a, the structural subunits and the native Hc, determined by fluorescence spectroscopy, coincide closely with the melting temperatures (T(m)), determined by CD spectroscopy. The free energy of stabilization in water, DeltaG(D)(H(2)O), determined from (Gdn. HCl) denaturation studies, is about two times higher for the structural subunit RtH1 and the whole hemocyanin molecule as compared to the functional unit RtH1-a. The oligomerization between the structural subunits or the eight functional units, assembled in subunit RtH1, has a stabilizing effect on the whole molecule as well as the structural subunits.     

Structure of hemocyanin subunit CaeSS2 of the crustacean Mediterranean crab Carcinus aestuarii

Arthropodan hemocyanins are giant respiratory proteins responsible for oxygen transport. They exhibit unusual assemblies of up to 48 structural subunits. Hemocyanin from Carcinus aestuarii contains three major and two minor structural subunits. Here, we reveal the primary structure of the gamma-type 75 kDa subunit of Carcinus aestuarii hemocyanin, CaeSS2, and combine structure-based sequence alignments, tryptophan fluorescence, and glycosylation analyses to provide insights into the structural and functional organisation of CaeSS2. We identify three functional domains and three conserved histidine residues that most likely participate in the formation of the copper active site in domain 2. Oxygen-binding ability of Carcinus aestuarii Hc and its structural subunit 2 was studied using CD and fluorescence spectroscopy. Removing the copper dioxygen system from the active site led to a decrease of the melting temperature, which can be explained by a stabilizing effect of the binding metal ion. To study the quenching effect of the active site copper ions in hemocyanins, the copper complex Cu(II)(PuPhPy)2+ was used, which appears as a very strong quencher of the tryptophan emission. Furthermore, the structural localization was clarified and found to explain the observed fluorescence behavior of the protein. Sugar analysis reveals that CaeSS2 is glycosylated, and oligosaccharide chains connected to three O-glycosylated and one N-glycosylated sites were found.     

Fluorescence properties and conformational stability of the beta-hemocyanin of Helix pomatia

The beta-hemocyanin (beta-HpH) is one of the three dioxygen-binding proteins found freely dissolved in the hemolymph of the gastropodan mollusc Helix pomatia. The didecameric molecule (molecular mass 9 MDa) is built up of only one type of subunits. The fluorescence properties of the oxygenated and apo-form (copper-deprived) of the didecamer and its subunits were characterized. Upon excitation of the hemocyanins at 295 or 280 nm, tryptophyl residues buried in the hydrophobic interior of the protein determine the fluorescence emission. This is confirmed by quenching experiments with acrylamide, cesium chloride and potassium iodide. The copper-dioxygen system at the binuclear active site quenches the tryptophan emission of the oxy-beta-HpH. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophyl residues in the apo-form. Time-resolved fluorescence measurements show that the oxygenated and copper-deprived forms of the beta-HpH and its subunits exist in different conformations. The thermal stability of the oxy- and apo-beta-HpH is characterized by a transition temperature (Tm) of 84 degrees C and 63 degrees C, respectively, obtained by differential scanning calorimetry. Increase of the temperature influences the active site at lower temperatures than the environments of tryptophans and tyrosines causing a loss of oxygen bound to the copper atoms. This process is, at least partially, reversible as after cooling of the protein samples, around 60% reinstatement of the copper-peroxide band has been observed. The results confirm the role of the copper-dioxygen complex for the stabilization of the hemocyanin structure in solution. The other important stabilizing factor is oligomerization of the hemocyanin molecule.     

Keyhole limpet hemocyanin type 2 (KLH2): detection and immunolocalization of a labile functional unit h

Keyhole limpet hemocyanin (KLH) is a mixture of two hemocyanin isoforms, termed KLH1 and KLH2. Within KLH1 eight oxygen-binding functional units (FUs), 1-a to 1-h, have been identified, in contrast to KLH2, which was previously thought to be organized in seven FUs (2-a to 2-g). By limited proteolysis of KLH2 subunits, isolation of the polypeptide fragments, and N-terminal sequencing, we have now identified an eighth FU of type h, with a molecular mass of 43 kDa. This is unusually small for a FU h from a gastropodan hemocyanin. It is also shown that KLH2 didecamers can be split into a stable and homogeneous population of decamers by dialysis against 50 mM Tris/HCl, pH 7.5, in the absence of divalent cations. Electron microscopic immunolocalization using a specific monoclonal antibody reveals that FU KLH2-h is located at the collar of the decamer.     

Fluorescence lifetime distributions in human superoxide dismutase. Effect of temperature and denaturation.

The internal dynamics of human superoxide dismutase has been studied using time-resolved fluorescence. The fluorescence decay has been analyzed using continuous distribution of lifetime values. The effect of temperature and conformational state on the lifetime distribution has been investigated. The emission of the single tryptophan residue depends on the nature and dynamics of the protein matrix. Conformational changes have been induced by increased concentration of guanidinium hydrochloride. We found that both temperature and conformation strongly effect the width of the lifetime distribution.     

Study of the biological significance of cytochrome methylation. I. Thermal, acid and guanidinium hydrochloride denaturations of baker's yeast ferricytochromes c.

The iso-cytochromes c from baker's yeast: iso-1 methylated and unmethylated forms and iso-2 have been purified and their stabilities towards denaturants compared to that of horse heart cytochrome c. Thermal, acid and guanidinium hydrochloride denaturations were followed using fluorescence emission of their tryptophan 59 and/or the absorbance in the Soret region as the physical parameters. Very few differences could be evidenced among the ferricytochromes investigated in this study insofar as the acid denaturations are concerned. This is to be contrasted with the conclusions of the thermal and guanidinium hydrochloride denaturations studies which clearly showed the ferricytochrome from horse heart to be much more stable than those from baker's yeast. No appreciable differences could be measured among the methylated and unmethylated forms of iso-1 cytochrome c nor among iso-1 and iso-2 cytochromes from baker's yeast. Our results suggest that a stabilizing effect of methylation on the tridimensional structure of ferricytochrome c must probably be discarded. Other possible physiological roles of methylation are suggested taking into account the relative instability of ascomycetes's cytochromes as compared to mammalian ones.     

Oligomeric stability of Rapana venosa hemocyanin (RvH) and its structural subunits

The two structural subunits RvH1 and RvH2 were separated after overnight dialysis of Rapana venosa Hc against 130 mM Gly/NaOH buffer, pH 9.6, on an ion exchange column Hiload 26/10 Sepharose Q using a fast performance liquid chromatography (FPLC) system. The reassociation characteristics of these two RvH isoforms and the native molecule were studied in buffers with different pH values and concentrations of Ca(2+) and Mg(2+). Reassociation of mixed RvH subunits was performed over a period of several days using a stabilizing buffer (SB) of pH 7.0 containing different concentrations of Ca(2+) and Mg(2+) ions. After 2 days of dialysis, an RvH subunit mixture of didecamers and multidecamers was observed in the presence of 100 mM CaCl(2) and MgCl(2), though RvH1 and RvH2 are biochemically and immunologically different and have also different dissociation properties. The reassociation, performed at pH 9.6 with 2 mM CaCl(2) and MgCl(2) at 4 degrees C over a period of one to several weeks, led to the formation of decameric oligomers, while didecamers formed predominantly in the SB at pH 7.0. Higher concentrations of calcium and magnesium ions led to a more rapid reassociation of RvH1 resulting in long stable multidecamers and helical tubules, which were stable and slowly dissociated into shorter multidecamers and decamers at higher pH values. The reassociation of the RvH2 structural subunit in the same buffers processed slowly and yielded didecamers, shorter tubule polymers and long multidecamers which are less stable at higher pH values. The stability of RvH isoforms under varying ionic conditions is compared with the stability of keyhole limpet (KLH, Megathura crenulata) hemocyanin (KLH) and Haliotis tuberculata hemocyanin (HtH) isoforms.The process of dissociation and reassociation is connected with changes of the fluorescence intensity at 600 nm, which can be explained by differences in opalescence of the solutions of these two isoforms. The solutions of longer tubule polymers and multidecamers of RvH1 show a higher opalescence compared to the solutions of shorter helical tubules and multidecamers of RvH2.     

Keyhole Limpet Hemocyanin Type 2 (KLH2): Detection and Immunolocalization of a Labile Functional Unit h

Keyhole limpet hemocyanin (KLH) is a mixture of two hemocyanin isoforms, termed KLH1 and KLH2. Within KLH1 eight oxygen-binding functional units (FUs), 1-a to 1-h, have been identified, in contrast to KLH2, which was previously thought to be organized in seven FUs (2-a to 2-g). By limited proteolysis of KLH2 subunits, isolation of the polypeptide fragments, and N-terminal sequencing, we have now identified an eighth FU of type h, with a molecular mass of 43 kDa. This is unusually small for a FU h from a gastropodan hemocyanin. It is also shown that KLH2 didecamers can be split into a stable and homogeneous population of decamers by dialysis against 50 mM Tris/HCl, pH 7.5, in the absence of divalent cations. Electron microscopic immunolocalization using a specific monoclonal antibody reveals that FU KLH2-h is located at the collar of the decamer.     

Origins of blood acetate in the rat.

1. Fluorimetric techniques were used to characterize the environment of tryptophan residues in thermolysin and apo-thermolysin. The apo-thermolysin was obtained by dissolving the enzyme in the presence of 10mm-EDTA, which removed the functional Zn(2+) ion and the four Ca(2+) ions/molecule from the enzyme. 2. At 25 degrees C in aqueous solution the fluorescence-emission spectrum of the native holoenzyme, on excitation at 290nm, was essentially characteristic of tryptophan, with an emission maximum at 333nm. The emission maximum of the apoenzyme is red-shifted to 338nm and the relative intensity of fluorescence is decreased by 10%, both effects indicating some unfolding of the protein molecule, with the indole groups being transferred to a more hydrophilic environment. 3. Fluorescence quenching studies using KI, N'-methylnicotinamide hydrochloride and acrylamide indicated a more open structure in the apoenzyme, with the tryptophan residues located in a negatively charged environment. 4. The thermal properties of the apoenzyme, as monitored by fluorescence-emission measurements, are dramatically changed with respect to the native holoenzyme. In fact, whereas the native enzyme is heat-stable up to about 80 degrees C, for the apoenzyme a thermal transition is observed near 48 degrees C. The apoenzyme is also unstable to the action of unfolding agents such as urea and guanidinium chloride, much as for other globular proteins from mesophilic organisms. 5. The functional Zn(2+) ion does not contribute noticeably to the stability of thermolysin. 6. It is concluded that a major role in the structural stability of thermolysin is played by the Ca(2+) ions, which have a bridging function within this disulphide-free protein molecule.     

Spectroscopic studies on the interaction of chromium (VI) and chromium (III) with keyhole limpet hemocyanin

The interactions of keyhole limpet hemocyanin (KLH) with chromium nitrate, potassium dichromate, and chromate were investigated using fluorescence, UV–vis absorption and circular dichroism (CD) spectroscopy under simulated physiological conditions. The experimental results showed that the different forms of chromium could quench the intrinsic fluorescence of KLH following a static quenching mechanism rather than by dynamic collision, which indicated that a Cr–KLH complex was formed. The Stern–Volmer quenching constants for the interaction indicated that the binding reaction of KLH with Cr(VI) was stronger the binding of KLH with Cr(III). The thermodynamic values for binding of Cr(VI) to KLH are ΔH > 0 and ΔS > 0. By contrast, the values for the interaction of Cr(III) with KLH are ΔH < 0 and ΔS < 0. The results of synchronous fluorescence, UV–vis absorption and CD spectroscopy showed that the α‐helical secondary structure and conformation of KLH were altered by different forms of chromium. Copyright © 2016 John Wiley & Sons, Ltd.     

Intrinsic tryptophan fluorescence of Schizosaccharomyces pombe mitochondrial F1-ATPase. A powerful probe for phosphate and nucleotide interactions

Mitochondrial F1 from the yeast Schizosaccharomyces pombe, in contrast to the mammalian enzyme, exhibits a characteristic intrinsic tryptophan fluorescence with a maximal excitation at 291 nm and a maximal emission at 332 nm. Low values of Stern-Volmer quenching constants, 4.0 M-1 or 1.8 M-1, respectively, in the presence of either acrylamide or iodide, indicate that tryptophans are mainly buried inside the native enzyme. Upon subunit dissociation and unfolding by 6 M guanidine hydrochloride (Gdn.HCl), the maximal emission is shifted to 354 nm, a value very similar to that obtained with N-acetyltryptophanamide, a solute-tryptophan model compound. The tryptophan content of each isolated subunit has been estimated by fluorescence titration in the presence of Gdn.HCl with free tryptophan as a standard. Two tryptophans and one tryptophan are found respectively in the alpha and epsilon subunits, whereas none is detected in the beta, gamma, and delta subunits. These subunit contents are consistent with the total of seven tryptophans estimated for native F1 with alpha 3 beta 3 gamma 1 delta 1 epsilon 1 stoichiometry. The maximal emission of the isolated epsilon subunit is markedly blue-shifted to 310-312 nm by interaction with the isolated delta subunit, which suggests that the epsilon subunit tryptophan might be a very minor contributor to the native F1 fluorescence measured at 332 nm. This fluorescence is very sensitive to phosphate, which produces a marked blue shift indicative of tryptophans in a more hydrophobic environment. On the other hand, ADP and ATP quench the maximal emission at 332 nm, lower tryptophan accessibility to acrylamide, and reveal tryptophan heterogeneity.     

p53 unfolding detected by CD but not by tryptophan fluorescence

Full-length human p53 protein was examined using tryptophan fluorescence and circular dichroism spectroscopy (CD) to monitor unfolding. No significant alteration in tryptophan fluorescence for the tetrameric protein was detectable over a wide range of either urea or guanidine hydrochloride concentrations, in contrast to results with the isolated DNA binding domain [Bullock et al. (1997) Proc. Natl. Acad. Sci. USA 94, 14338]. Under similar denaturant conditions, CD demonstrated significant protein unfolding for the full-length wild-type protein, with increased apparent structure loss compared to that detected during thermal denaturation [Nichols and Matthews (2001) Biochemistry 40, 3847]. Examination of X-ray structures containing two of the four tryptophan residues of a p53 monomer suggested local environments consistent with quenched fluorophores. Exploration of p53 fluorescence using potassium iodide as a quencher confirmed that these fluorophores are already substantially quenched in the native structure, and this quenching is not relieved during protein unfolding.     

Structure-based calculation of multi-donor multi-acceptor fluorescence resonance energy transfer in the 4×6-mer tarantula hemocyanin

Hemocyanins are oxygen carriers of arthropods and molluscs. The oxygen is bound between two copper ions, forming a Cu(II)-O₂ ^2–-Cu(II) complex. The oxygenated active sites create two spectroscopic signals indicating the oxygen load of the hemocyanins: first, an absorption band at 340 nm which is due to a ligand-to-metal charge transfer complex, and second, a strong quenching of the intrinsic tryptophan fluorescence, the cause of which has not been definitively identified. We showed for the 4×6-mer hemocyanin of the tarantula Eurypelma californicum that the fluorescence quenching of oxygenated hemocyanin is caused exclusively by fluorescence resonance energy transfer (FRET). The tarantula hemocyanin consists of 24 subunits containing 148 tryptophans acting as donors and 24 active sites as acceptors. The donor–acceptor distances are determined on the basis of a closely related crystal structure of the horseshoe crab Limulus polyphemus hemocyanin subunit II (68–79% homology). Calculation of the expected fluorescence quenching and the measured transfer efficiency coincided extraordinary well, so that the fluorescence quenching of oxygenated tarantula hemocyanin can be completely explained by Förster transfer. This results explain for the first time, on a molecular basis, why fluorescence quantum yield can be used as an intrinsic signal for oxygen load of at least one arthropod hemocyanin, in particular that from the tarantula.     

Long-lived tryptophan fluorescence in phosphoglycerate mutase

Phosphoglycerate mutase (PGM; EC 2.7.5.3) isolated from rat and rabbit muscle has been shown to possess an unusually long-lived fluorescence component when excited by ultraviolet light below 310 nm. On the basis of spectral and physical measurements, this 16.4 (+/- 0.2) ns fluorescence lifetime at room temperature is assigned to a tryptophan residue in an unusual environment. The emission profile of this long-lived tryptophan is red shifted from the other tryptophans of PGM by approximately 25 nm. PGM has been crystallized and sequenced from yeast where it has been shown to be a tetramer with 29K subunits. However, we have not been able to detect the existence of an unusually long-lived fluorescence component in the yeast isomer. The long fluorescence lifetime is lost upon denaturation of rabbit PGM and is partially restored upon introduction of the protein to a nondenaturing environment, suggesting the long lifetime is not the result of a covalent modification. The PGM molecule was studied by a number of techniques including time-resolved tryptophan fluorescence, quenching studies of tryptophan fluorescence, and enzyme activity studies. The long-lived fluorescence has been shown to be statistically quenched by Br-, I-, and Cu2+ in the submillimolar region while the acrylamide quenching shows the tryptophan is marginally accessible to solvent. Characterization of the long-lived fluorescence and its possible sources are discussed.     

The role of tryptophan residues in the antigenic activity of carcinoembryonic antigen

Antigenic determinants of carcino-embryonic antigen (CEA) were spatially located using N-bromosuccinimide modification of tryptophan residues both in native (acetate buffer solution) and unfolded (guanidinium chloride solution) molecule of the antigen. Modification of exposed tryptophan residues failed to alter CEA antigenic activity and conformation of its protein portion as shown by CD spectroscopy. On the contrary, modification of buried tryptophan residues induced conformational changes of CEA protein portion connected with a considerable loss of its antigenic activity. It was shown that CEA antigenic activity depends on spatial structure of its protein moiety.     

Primary structure and unusual carbohydrate moiety of functional unit 2-c of keyhole limpet hemocyanin (KLH)

The complete amino acid sequence of the Megathura crenulata hemocyanin functional unit KLH2-c was determined by direct sequencing and matrix-assisted laser desorption ionization mass spectrometry of the protein, and of peptides obtained by cleavage with EndoLysC proteinase, chymotrypsin and cyanogen bromide. This is the first complete primary structure of a functional unit c from a gastropod hemocyanin. KLH2-c consists of 420 amino acid residues. Circular dichroism spectra indicated approx. 31% beta-sheet and 29% alpha-helix contents. A multiple sequence alignment with other molluscan hemocyanin functional units revealed average identities between 41 and 49%, but 55% in case of Octopus hemocyanin functional unit c which is the structural equivalent to KLH2-c. KLH2-c has a molecular mass of approx. 48 kDa as calculated from its sequence and a measured mass of approx. 56 kDa; the mass difference is attributed to the sugar side chains usually decorating molluscan hemocyanin. However, inspection of the sequence of KLH2-c revealed no potential N-linked carbohydrate attachment sites, and this was supported by its inability to bind concanavalin A. Also KLH1-c was unreactive, whereas most, if not all, other functional units of KLH1 and KLH2 reacted positively to this lectin. On the other hand, peanut agglutinin specifically binds KLH2-c, indicating the presence of O-glycosidically linked carbohydrates in this functional unit. This contrasts to all other KLH functional units (including KLH1-c), which lack O-linked glycosides. The present results are discussed in view of the recent X-ray structure of the functional unit g from Octopus hemocyanin, and a published record of the Thomsen Friedenreich tumor antigenic epitope in KLH.     

The effect of metals on SDS-induced partially folded states of CopC

In this work, the unfolding of CopC was used to elucidate details of the protein structure through different spectroscopic techniques. The interactions of CopC and its mutants with the anionic surfactant sodium dodecyl sulfate (SDS), guanidinium hydrochloride, and urea were monitored by fluorescence spectroscopy, far-UV circular dichroism spectroscopy, and fluorescence lifetime measurements. The interaction of SDS with CopC resulted in the formation of a partially folded intermediate. In this intermediate, the structure of the C-terminal is unfolded, whereas the N-terminal retains the native structure. Further, we have explored the effects of metals on the intermediate in aqueous surfactant. The results suggested that the Ag⁺ ion has a large effect on the unfolding induced by SDS. In addition, the binding capacity of the different unfolding degree protein toward Cu²⁺ indicated the high stability of the N-terminal. The protein–Cu²⁺ unfolding induced by guanidinium hydrochloride and urea caused the binding of Cu²⁺ to increase the stability of the N-terminal, which resulted in an intermediate in the unfolding process. The first transition corresponded to unfolding of the C-terminal, and the second transition was attributed to unfolding of the N-terminal. Furthermore, the anisotropy decay indicated that the motion of tryptophan occurred at a higher urea concentration, which suggested the high stability of the N-terminal. Steered molecular dynamics simulations also indicated that the structure of the N-terminal was rigid.     

Conformational properties of human immunoglobulin G subclasses analysis by temperature-perturbation and solvent-perturbation spectroscopy

Conformational properties of human myeloma immunoglobulins G belonging to four subclasses (IgG1 Van, IgG2 Kom, IgG3 Pla, IgG4 Ang), and also Fab, Fc and pFc′ fragments derived from IgG1 Van, IgG2 Kom and IgG3 Pla have been studied by temperature-perturbation and solvent-perturbation spectroscopy. It has been shown that the immunoglobulins studied practically do not differ in the number of tyrosine and tryptophan residues exposed to different solvent perturbants (saccharose, glycerol, dimethylsulfoxide). The same regularity is observed for isolated Fab and Fc fragments. At the same time, the immunoglobulins compared and their proteolytic fragments significantly differ in the number of aromatic chromophores perturbed by temperature. These data indicate that immunoglobulins of different subclasses and their subunits have a different rigidity of structure in relation to thermal perturbation. The Fc subunits of IgG1 are characterized by the lowest rigidity of structure of internal hydrophobic cores of domains (characterized by the rigidity of the microenvironment of tryptophan residues), as compared with the Fc subunits of IgG2 and IgG3. In the case of IgG1 and IgG2, these differences seem to be brought about by a different rigidity of structure of C_H2 domains, since thermal-perturbation spectra of the pFc′ fragments of these subclasses practically coincide. The total number of chromophores exposed to different solvent perturbants in the isolated Fab and Fc fragments practically coincides with the number of exposed chromophores in intact immunoglobulins. Similar coincidence is observed for the tryptophan residues perturbed by temperature. These data indicate that neither the conformation of surface sites nor the conformation of internal hydrophobic cores of domains significantly changes on isolation of Fab and Fc fragments. At the same time, many more tyrosine residues are perturbed by temperature in the intact immunoglobulin G1 Van than in the corresponding sum of isolated Fab and Fc fragments, while for IgG2 Kom, which has the same length of hinge region, these values practically coincide. This fact can be explained by the greater temperature dependence of motions of subunits in IgG1 Van as compared with IgG2 Kom, and as a result of this by the higher mutual temperature-dependent influence of subunits on their internal structure (on interdomain interactions).     

Evidence of stable monomeric species in the unfolding of Cu,Zn superoxide dismutase from Photobacterium leiognathi.

The equilibrium unfolding process of Photobacterium leiognathi Cu,Zn superoxide dismutase has been quantitatively monitored through circular dichroism (CD) and fluorescence spectroscopy, upon increasing the guanidinium hydrochloride concentration. The study has been undertaken for both the holo- and the copper-free derivative to work out the role of copper in protein stability. In both cases the unfolding was reversible. The denaturation curve derived from CD and fluorescence spectroscopy was not coincident, suggesting that the denaturation process occurs through a three-state model with formation of an intermediate monomeric species. The occurrence of an intermediate species has been unambiguously demonstrated following CD and steady-state fluorescence spectra of the enzyme at various concentrations in presence of a fixed amounts of guanidinium hydrochloride.     

The luminescence properties of concanavalin A.

1. The luminescence properties of native concanavalin A, both at room temperature and at 77 degrees K, are similar to those of other proteins containing tyrosine and tryptophan. 2. Binding of methyl alpha-D-glucopyranoside to concanavalin A causes a slight reduction of its fluorescence at room temperature. 3. Removal of Mn2+ and Ca2+ ions from concanavalin A causes a small increase in its fluoresence. The fluorescence: phosphorescence ratio and phosphorescence lifetime of apo-concanavalin A are similar to those of tryptophan. 4. Denaturation of concanavalin A by urea and by guanidine hydrochloride apparently takes place in two stages. Apo-concanavalin A is more easily denatured than the native molecule, but concavalin A combined with methyl alpha-D-glucopyranoside is more resistant to denaturation. 5. The luminescence properties of concanavalin A are pH-dependent. 6. The results have been interpreted in terms of the known structure and properties of concanavalin A.