Red Shift Of Emission Maximum Research Articles

Allophycocyanin from Nostoc sp. phycobilisomes. Properties and amino acid sequence at the NH2 terminus of the alpha and beta subunits of allophycocyanins I, II, and III.

Allophycocyanin from Nostoc sp. phycobilisomes was separated into four spectrally distinct components designated allophycocyanin I, B, II, and III by adsorption chromatography on brushite columns. Allophycocyanins I and B had red-shifted fluorescence emission maxima, and on this basis, may function in transfer of excitation energy from phycobilisomes to chlorophyll a. Allophycyanins II and III, which together comprise 70% of the total allophycocyanin, have absorption maxima at 648 nm and 650 nm, respectively, and probably transfer excitation energy from phycocyanin to allophycocyanins I and B, in addition to serving a structural function. Allophycocyanin I was resolved into alpha, beta, and gamma subunits with apparent molecular weights of 18,000, 17,000, and 35,000, respectively, whereas allophycocyanin B was resolved into two subunits with apparent molecular weights of 16,100 and 15,300, using a modified Weber and Osborn gel electrophoresis system (Brown, A. S., and Troxler, R. F. (1977) Biochem. J. 163, 571-581). In the same gel system, allophycocyanins II and III were each resolved into alpha and beta subunits with apparent molecular weights of 18,000 and 17,000, respectively. The subunits of allophycocyanins I, II, and III were isolated by gel filtration and ion exchange chromatography and the amino acid compositions determined. Automated sequence analysis demonstrated that the first 30 amino acids at the NH2 terminus of alpha subunits, and the beta subunits, of allophycocyanins I to III were identical. alpha Subunits: Ser-Ile-Val-Thr-Lys-Ser-Ile-Val-Asn-Ala-Asp-Ala-Glu-Ala-Arg-Tyr-Leu-Ser-Pro-Gly -Glu-Leu-Asp-Arg-Ile-Lys-Ser-Phe-Val-Thr- beta Subunits: Ala-Gln-Asp-Ala-Ile-Thr-Ala-Val-Ile-Asn-Ala-Ala-Asp-Val-Gln-Gly-Lys-Tyr-Leu-Asp-Ala-Thr-Ala-Leu-Ser-Lys-Leu-Lys-Ala-Tyr- The gamma subunit of allophycocyanin I and both subunits of allophycocyanin B appeared to be blocked at the NH2 terminus, suggesting that the allophycocyanin B subunits may be different gene products than those of allophycocyanins I to III, or if the same, the subunits of allophycocyanin B undergo proteolytic modification after initial synthesis.

The interaction of human serum albumin and hemopexin with porphyrins

The interactions of the porphyrin-binding proteins of human serum, albumin and hemopexin, with coproporphyrin I and III, uroporphyrin I, protoporphyrin IX and zinc-protoporphyrin IX were examined quantitatively by means of equilibrium dialysis and fluorescence. Albumin bound the porphyrins with a high affinity, dissociation constant ( K d ) in the μM range, with the exception of uroporphyrin I which was only weakly bound, K d of the order of 100 μM. Protoporphyrin IX and zinc-protoporphyrin IX were more strongly bound than coproporphyrin I and III suggesting common binding sites. Human hemopexin formed complexes with coproporphyrin I and III, K d near 0.5 μM, with protoporphyrin IX, K d near 2 μM, and with zinc-protoporphyrin IX, K d near 0.5 μM. All porphyrins were bound with an apparent stoichiometry of 1 : 1, except zinc-protoporphyrin, 2 mol of which appear to be bound per mol of hemopexin. As with albumin, only a weak interaction with uroporphyrin I is evident, K d near 50 μM. Formation of the heme-hemopexin complex almost completely abolished subsequent interaction with coproporphyrin, consistent with heme and porphyrins sharing the same primary binding site on hemopexin. Several types of competition experiment were conducted to test directly the relative affinities of the two proteins for these porphyrins. Under these conditions, albumin clearly has a higher affinity than hemopexin for both protoporphyrin and zinc-protoporphyrin. Hemopexin is able to compete effectively with albumin for coproporphyrin I or III only at low molar ratios of the two proteins and seems unlikely to bind a significant portion of these porphyrins at the molar ratios (about 1 : 40) of hemopexin and albumin found in human serum. Further evidence for species differences in the heme binding site of hemopexin was obtained from fluorescence measurements on the human and rabbit proteins. Firstly, human hemopexin displayed a higher apparent stoichiometry of zinc-protoporphyrin binding (1.9 : 1) than rabbit hemopexin (1.3 : 1), with zinc-protoporphyrin-human hemopexin exhibiting a slightly higher relative fluorescence at 595 nm. Secondly, protoporphyrin bound to human hemopexin had red-shifted excitation (410 vs. 407 nm) and emission (634 vs. 627 nm) maxima compared with rabbit hemopexin. Thirdly, coproporphyrin I produced little quenching of the human protein compared with that of the rabbit protein.

Subunit interactions of rabbit muscle aldolase. Conformational change of aldolase in magnesium chloride and its relationship to the dissociation of subunits

Conformational changes of aldolase in MgCl2 have been studied with difference spectrophotometry, spectrophotofluorometry and circular dichroism. The difference spectra observed in MgCl2 (0·3 to 3·2 m) are characterized by negative absorption peaks at 293, 287 and 280 nm suggesting a change in the environment of tyrosine and tryptophan residues. Fluorescence studies show that a small but significant quenching occurred at 0·3 to 1·2 m-MgCl2 but enhanced fluorescence with a 5-nm red shift of emission maximum was observed at 1·5 to 3·2 m-MgCl2. The circular dichroism of aldolase [ϑ]220, of aldolase decreased 20% between 1·5 and 2·1 m-MgCl2. The transition profiles of the optical parameters indicate that the conformational change of aldolase in MgCl2 is not compatible with a two-state (native and post-transition state) transition mechanism. An intermediate conformational state appears to exist at 1·2 to 1·5 m-MgCl2 which is distinguishable from the native state only in the difference in the local environments of four to six residues of tyrosine and one to two residues of tryptophan and a decrease in Vmax towards fructose-1,6 bisphosphate. Above 2·8 m-MgCl2 a post-transitional state appears to exist which is characterized by 20%-less helix than native aldolase, exposure of about 90% of the tyrosine and about 80% of the tryptophan residues, and incorporation of three or four tryptophan residues into newly organized structural regions with enhanced fluorescence emission. The spectroscopic data obeyed a linear relationship with protein concentration at 0·9 m to 2·4 m-MgCl2 despite the increased subunit dissociation with a lowering of protein concentration that has been observed (Hsu # Neet, 1973) , indicating that the conformation of the monomer subunit is essentially identical to that of the monomeric unit when in the tetramer.

Fluorimetric and spectrophotometric studies of DPN-linked isocitrate dehydrogenase from bovine heart. Properties of tyrosyl and tryptophyl residues

The emission maximum of DPN-linked isocitrate dehydrogenase in pH 7.07 buffer is shifted from 317 to 324 nm and fluorescence intensity is decreased when the excitation wave-length is varied from 270 to 290 nm; in 0.2 M KOH, where the fluorescence of tyrosyl residues is almost completely quenched, a further substantial decline in quantum yield of protein fluorescence and a red shift of the emission peak to 339 nm occur. The latter should be due mainly to tryptophyl residues. The enzyme contains 9.4 tyrosyl residues per subunit of molecular weight 42,000 determined spectrophotometrically (295 nm) at pH 13, in good agreement with a tyrosine content of 9.7 by amino acid analysis. No more than 1.1 tyrosyl residues per subunit can be detected up to pH 10.6 at 7 degrees upon prolonged incubation. The increase in absorption at 295 nm with increasing pH is related to loss of enzyme activity and results in a red shift of the emission maximum, and decreased fluorescence intensity. Treatment of the enzyme in a Li+-containing buffer at pH 7.5 with an excess of N-acetylimidazole results in (a) modification of 1.1 tyrosyl residues per subunit, (b) a 30% decrease in enzyme activity, (c) a 6-nm red shift in emission maximum, and (d) a decrease in fluorescence intensity. Manganous DL-isocitrate (1.06 mM) prevents the acetylation of the enzyme. Deacetylation of the O-acetylated enzyme by hydroxylamine completely restores the enzyme activity and reverses the spectral changes. The acetylation studies indicate that the reactive tyrosyl residue does not participate directly in catalysis but may be involved in maintaining the proper conformation of the active enzyme center. A net of 1 of the 2 tryptophyl residues per subunit is perturbed immediately by a number of solvents. This perturbation is not affected by manganous isocitrate, whereas exposure of tyrosyl residues occurs only with time and is prevented by the substrate. The perturbation of the tryptophyl residue is accompanied by a red shift of the fluorescence emission maximum. The more exposed tryptophyl residue may contribute to the energy transfer from protein to nucleotides since the quenching of protein fluorescence upon binding of DPN+, DPNH, or ADP by enzyme results in a blue shift of the emission maximum. Manganous DL-isocitrate (1.06 mM) quenches protein fluorescence by 16% without a shift in emission peak and does not affect the relative extent of fluorescence quenching induced by the nucleotides.