Chromophore formation

Reid, B. G., and Flynn, G. C. (1997). Chromophore formation in green fluorescent protein. Biochemistry 36 6786-6791. 

Sniegowski JA, Lappe JW, Patel HN, Huffman HA, Wachter RM (2005) Base catalysis of chromophore formation in Arg96 and Glu222 variants of green fluorescent protein. J Biol Chem 280 26248-26255... 

Verkhusha W, Chudakov DM, Gurskaya NG, Lukyanov S, Lukyanov KA (2004) Common pathway for the red chromophore formation in fluorescent proteins and chromoproteins. Chem Biol 11 845-854... 

Fig. 5.2. Chromophore formation in avGFP and DsRed. Chromophore formation in avGFP (A) requires folding of the tripeptide into the right conformation in order to enable cyclization and oxidation to form the mature green chromophore. In DsRed (B) chromophore formation follows the same path as for avGFP but requires an additional oxidation step to extend the conjugation of the chromophore.

The steps leading to the formation of the intrinsic chro-mophore have recently been investigated kinetically with S65T-GFP. The process of chromophore formation is an ordered sequence of three distinct steps (1) slow protein folding (kf = 2.44 X 10 s ) that precedes chromophore modification (2) an intermediate step occurs that includes, but may not be necessarily limited to, cycli-zation of the tripeptide chromophore motif (kc = 3.8 X 10 s ) and (3) rate-limiting oxidation of the cyclized chromophore (kox = 1 51 X s ). Reid and Flynn also reasoned that because chromophore forms de novo from purified denatured protein and is a first-order process, GFP chromophore formation is likely to be an autocatalytic process. 

Condensation reactions of lignin have been mentioned 22) in connection with sulfite pulping as being responsible for color formation. However, most of the likely condensation reactions occurring during a kraft cook do not lead directly to chromophore formation. One potential condensation reaction which would give a chromophore is the formation of a chalcone (XIX) from vanillin and acetoguaiacone, both of which have been isolated by Enkvist 12) from black liquor. 

Schmidt, J.A., Heitner, C., "Light-Induced Yellowing of Mechanical and Ultra-High Yield Pulps. Part I. Effect of Methylation, NaBH Reduction and Ascorbic Acid on Chromophore Formation , J. Wood Chem. Technol., 1991,11(4) 397. 

Ideally, dissolution of cellulose in the amine N-oxide is supposed to be an entirely physical process without any chemical changes of pulp or solvent. However, in real-world processes there are several chemical processes observed, which cause formation of appreciable amounts of byproducts. A strong discoloration of the solution due to chromophore formation has been observed, which is accompanied by degradation of both the solute cellulose and the solvent NMMO at the elevated process temperatures, which in turn can provoke very severe effects, such as degradation of cellulose, temporary or permanent discoloration of the resulting fibers, decreased product... 

Colorometric Studies. None of the solution components of the o-dianisidine activity assay interfere with the detection of chromophore formation at 500 nm. The UV/visible spectrum of PEUU (on Mylar backing) is approximately flat in the vicinity of 500 nm, indicating that PEUU should not interfere with the assay. Inclusion of PEUU in the reference cuvette further reduced the possibility of interference from PEUU. 

A free radical attack upon the nitrile group was detected by Brandrup et al. o.si) In an investigation towards the processes responsible for chromophore formation in PAN, it was found that PAN, obtained from a normal free radical polymerization, always contains a certain ammint of enamines and p-ketonitriles. Their formation was ascribed to the following reaction sequence ... 

Peroxide was the original oxidant, but its incomplete removal quenched the chromophore reaction. Stegemann introduced the oxidant Chlora-mine-T, which has been widely adopted because it is simple to neutralize and does not interfere with chromophore formation (S22). Using Chlor-... 

FIGURE 3.3 Chromophore formation from phenyl courmaran model compounds. Acidolysis gives a phenyl coumarone alkaline hydrolysis gives a stilbene. 

JA Schmidt, C Heitner. Light-induced yellowing of mechanical and ultrahigh yield pulps. Part 1. Effect of methylation, sodium borohydride reduction and ascorbic acid on chromophore formation. JWood Chem Technol 11(4) 397 18, 1991. 

Gierer and SY Lin. Photodegradation of Lignin A Contribution to the Mechanism of Chromophore Formation. Svensk Papperstidn 75 233-239, 1971. 

GFP from the jellyfish Aequorea victoria has 238 residues, which form 11 / -sheets arranged in a barrel shape (Fig. 5.2). The barrel is a nearly perfect cylinder with a height of 42 A and a radius of 12 A. The chromophore is located in the middle of the so-called / -barrel and deletion mapping experiments have shown that nearly the entire structure (residues 2-232) is required for chromophore formation and/or fluorescence [18]. 

Scheme 5.1 Chromophore formation in green fluorescent protein.

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