Spectroscopic properties of tetrapyrroles on denatured biliproteins

Guard-Friar, Deborah; MacColl, Robert

doi:10.1016/0003-9861(84)90111-5

Cited by 11 publications

(9 citation statements)

References 30 publications

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“…They were then tested against selected biliproteins from blue-green (Cphycocyanin and C-phycoerythrin) and red algae (B-phycoerythrin). EXPERIMENTAL Biliproteins were isolated and purified from crytomonads, phycocyanin 612-Hemiselmis virescens, phycocyanin 645-Chroomonas species, and phycoerythrin 545-Rhodomonas lens, and phycoerythrin 566-Cryptomonas ovata, as described previously (14). The cryptomonad proteins were extensively purified by (NH4)2SO4 fractionation and gel filtration on Sepharose 4B and Ultrogel AcA54.…”

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“…Its immunochemistry is thus clearly distinctive from C-phycocyanin and R-phycocyanin to which it has some spectral resemblance. In addition to these immunochemical studies on phycocyanin 612, recently its biochemical and spectroscopic properties were evaluated (14,17). Its chromophore content is unique and differs from that of C-phycocyanin in that its ,B subunit has a cryptoviolin chromophore in addition to the usual two phycocyanobilins.…”

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“…Its a subunit, however, has the same chromophore content as C-phycocyanin but differs from that found for cryptomonad phycocyanin 645 (15,18). A common feature in the chromophore contents of cryptomonad biliproteins is that cryptoviolin is always present ( 14,(17)(18)(19).…”

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Immunochemistry on Cryptomonad Biliproteins

Guard-Friar¹,

Eisenberg²,

Edwards³

et al. 1986

Plant Physiol.

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ABSTRACIA survey is made of the immunochemical behavior of four of the six known types of cryptomonad biliproteins: phycocyanins 612 and 645 and phycoerythrins 545 and 566. They were compared both among themselves and to selected biliproteins isolated from blue-green and red algae. All the cryptomonad biliproteins were shown to be closely related to each other by Ouchterlony double diffusion technics. An antigenic relationship among all the cryptomonad biliproteins and B-phycoerythrin (red alp) and C-phycoerythrin (blue-green alga) was established. Only a very marginal cross-reactivity was found between C-phycocyanin (blue-green algae) and the cryptomonad biliproteins. These results suggest a common ancestor for the photosynthetic units of all three biliprotein-containing phyla.Biliproteins are light harvesting and excitation energy transfer chromoproteins that function as accessory pigments for PSII in the photosynthesis of blue-green (cyanobacterial), red, and cryptomonad algae. Cryptomonad biliproteins are unusual in that they apparently do not form phycobilisomes and in the cryptomonads no analog of allophycocyanin has yet been detected. There are six biliproteins in the cryptomonads, phycocyanins 612, 630, 645, and phycoerythrins 545, 555, 566, with only one type usually found in each alga.Immunochemical studies on biliproteins have greatly emphasized blue-green and red algal examples. By 1967 four publications had established in a very extensive and decisive manner three basic rules governing the immunochemical behavior of these two types of algal biliproteins (1-3, 29). These three generalizations are: all phycocyanins from both blue-green (procaryotes) or red (eucaryotes) algae are immunochemically very similar; all phycoerythrins of all spectral types (C-, R-, B-) are immunochemically closely related whether they are from bluegreen or red algae; no phycocyanin is related immunochemically to any phycoerythrin. This last rule holds even when the phycocyanin and phycoerythrin are isolated from the same alga. These first two results were most important because of the great differences in structure between procaryotic blue-green algae and eucaryotic red algae. The immunochemical results then established the principle that, although major cellular changes have occurred in the evolution from procaryotes to eucaryotes, the individual proteins could remain virtually unaltered. Subsequent research has shown that the two subunits of phycoerythrin are immunochemically related (27) Research on the immunochemistry of cryptomonad biliproteins, has been much less extensive and then usually only a fragment of a larger study. It is therefore not too surprising that unlike the comparison of blue-green and red algal biliproteins, cryptomonad results have been so far perhaps inconclusive and controversial (2,4,12,13,16,26,29). We, therefore, undertook an immunochemical investigation using Ouchterlony doublediffusion and four different cryptomonad biliproteins. Two different phycoerythrins and two different phycocyanins ...

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Immunochemistry on Cryptomonad Biliproteins

Guard-Friar¹,

Eisenberg²,

Edwards³

et al. 1986

Plant Physiol.

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“…As shown in Figure 1B, we find three significant transition points in our data: a sharp decrease at approximately 0.1 M urea, a second decrease at 1.1 M urea and third transition at 5.4 M. The transition that is the easiest to understand and assign is the large transition at 5.4 M; it is well documented in the literature that the sharp decrease in visible absorption at high urea concentrations is due to loss of the heterodimeric unit (αβ) coupled with unfolding of the tertiary structure (15,21,29). The tertiary structure of the protein is no longer working as a scaffold to hold the tetrapyrrole chromophore in a linear conformation, and the chromophore assumes a 'lockwasher' conformation with a much smaller molar absorptivity in the visible region (9,30).…”

Section: Relationship Between the Absorption Spectrum And The Oligomementioning

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The Free Energy of Dissociation of Oligomeric Structure in Phycocyanin Is Not Linear with Denaturant

et al. 2006

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Using SEC HPLC and fluorescence anisotropy, absorption spectra were assigned to the specific oligomeric structures found with phycocyanin. The absorption spectra were used to quantify the population of each oligomeric form of the protein as a function of both urea concentration and temperature. Phycocyanin hexamer dissociate to trimers with equilibrium constants of 10 −6 to 10 −5 . Phycocyanin trimers dissociate to monomers with equilibrium constants of 10 −15 to 10 −12 . Both dissociation constants increase linearly with increasing urea concentration, and ΔG° values calculated from the equilibrium constants fit best with an exponential function. Our findings appear in contrast with the commonly used linear extrapolation model, ΔG urea° = ΔG water° + A[denaturant], in which a linear relationship exists between the free energy of protein unfolding or loss of quaternary structure and the denaturant concentration. Our data examines a smaller range of denaturant concentration than generally used which might partially explain the inconsistency.Many active biological complexes are formed from the association of small subunits. While large proteins should be more stable due to their increased ratio of buried to solvent-exposed surface area, a multidomain protein folds more slowly than a single domain protein (1-3). Thus, there appear to be some advantages to building active complexes through small, single domain subunits. There is a broad and detailed body of knowledge on both the kinetics and thermodynamics of single domain folding, but the field of knowledge for the association or oligomerization process is more limited.In this paper we examine the thermodynamics of the urea induced dissociation of the oligomeric structures in phycocyanin. Phycocyanin is a chromoprotein found in the light harvesting antenna or phycobilisome of cyanobacteria (4,5). The basic unit of phycocyanin is a heterodimeric αβ; the 17.4 kD α subunit contains one covalently bound chromophore while the 17.4 kD β subunit contains two covalently bound chromophores. The chromophore is a conjugated tetrapyrrole system known as the phycocyanobilin. There is large degree of homology between the α and β subunits with each subunit having an all-helical globin-like structure. The protein used in this study has been isolated from Agmenellum quadruplicatum, and the crystal structure of hexameric phycocyanin isolated from this species has been determined (6,7).

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“…This separation establishes that the a and p subunits are not joined by disulfide bonds. In addition it has recently been shown that mercaptoethanol can produce spurious results in the calculation of the chromophore contents of these biliproteins (Guard-Friar and MacColl, 1984). The mercaptoethanol-free experiments allow analysis of the chromophore content in a rapid and artifact-free manner.…”

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SUBUNIT SEPARATION (α, αprime;, β) OF CRYPTOMONAD BILIPROTEINS *

Guard-Friar¹,

MacColl²

1986

Photochem & Photobiology

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Abstract— Phycocyanin 645, phycocyanin 612 and phycoerythrin 545 are biliproteins isolated from the cryptomonad algae, Chroornonas species, Hemiselmis virescens and Rhodomonas lens, respectively. The protein has α and β subunits. which are separated on an ion exchange column by using a urea gradient and omitting 2‐mercaptoethanol from the solvent. This separation establishes that the α and β subunits are not joined by disulfide bonds. In addition it has recently been shown that mercaptoethanol can produce spurious results in the calculation of the chromophore contents of these biliproteins (Guard‐Friar and MacColl, 1984). The mercaptoethanol‐free experiments allow analysis of the chromophore content in a rapid and artifact‐free manner. When the ion exchange chromatography of phycocyanin 645 is manipulated by changing the type of urea gradient, two distinct a subunit fractions are obtained. These two fractions have identical visible absorption spectra but different amino acid compositions. At least two different gene products are, thus. responsible for the a subunits. The sole chromophore on the two a subunits of phycocyanin 645 is the unique 697‐nm bilin as seen in acidic urea. Its reactivity with mercaptoethanol is determined. The α subunits of phycoerythrin 545 have two different bilins: cryptoviolin and phycoerythrobilin. Phycocyanobilin is the chromophore on the α subunit of phycocyanin 612.

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Spectroscopic properties of tetrapyrroles on denatured biliproteins

Cited by 11 publications

References 30 publications

Immunochemistry on Cryptomonad Biliproteins

Immunochemistry on Cryptomonad Biliproteins

The Free Energy of Dissociation of Oligomeric Structure in Phycocyanin Is Not Linear with Denaturant

SUBUNIT SEPARATION (α, αprime;, β) OF CRYPTOMONAD BILIPROTEINS *

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