DL-tryptophan amide hydrochloride

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.

Keyhole limpet hemocyanin is a respiratory glycoprotein of high molecular weight from the gastropod mollusc Megathura crenulata. Two subunits, KLH1 and KLH2, were isolated using ion exchange chromatography and their physical properties are compared with the parent molecule. The various proteins are characterized by fluorescence spectroscopy, combined with fluorescence quenching studies, using acrylamide, cesium chloride and potassium iodide as tryptophan quenchers. The conformational stability of the native aggregate and its isolated structural subunits are also studied by circular dichroism and fluorescence spectroscopy as a function of temperature, as well as in the presence of guanidinium hydrochloride and urea. The associated subunits in the hemocyanin aggregates increase considerably the melting temperature to 67 degrees C and the free energy of stabilization in water, DeltaG(H(2)O)(D), towards guanidinium hydrochloride is higher for the decamer as compared to the isolated subunits; this difference can be accounted for by the stabilizing effects of intra-subunit interactions exerted within the oligomer. The copper-dioxygen complex at the active site additionally stabilizes the molecule, and removing of the copper ions increases the tryptophan emission and the quantum yield of the fluorescence.

MLGL (Mulberry Latex Galactose-specific Lectin) is an α-d-galactose binding lectin isolated from the latex of mulberry (Morus indica) tree and contains two tryptophan residues in each of its subunits. The fluorescence emission maximum of native MLGL seen at 326nm shifts to 350nm upon incubation with 6M guanidinium thiocyanate (Gdn.SCN), suggesting that the tryptophans are located inside the hydrophobic core of the protein and become fully exposed upon denaturation. Fluorescence quenching studies revealed that the neutral acrylamide exhibits the highest quenching, with ~33% of total fluorescence in the native protein being quenched at a quencher concentration of 0.5M, whereas iodide (~24%) and cesium (~4%) ions showed significantly lower quenching. With the denatured protein, acrylamide quenching involves both dynamic and static processes as evident from an upward curving Stern-Volmer plot. Time-resolved fluorescence studies showed two lifetime components of 3.7ns and 1.3ns for the native protein, while three lifetime components were observed for the denatured protein. MLGL showed high resistance to urea (up to 8M) and guanidine hydrochloride (up to 6M), whereas treatment with 6M Gdn.SCN completely denatured the protein, via a broad sigmoidal transition with a transition midpoint at ~3.75M. Circular dichroism studies and hemagglutination assays showed that the secondary and tertiary structures as well as lectin activity of MLGL were unaffected up to 70°C. Additionally, pH dependent studies showed that the secondary structure of MLGL is unaltered in the pH range 6.2 to 8.5, but a decrease in lectin activity is observed (~50%) at pH6.2.