FRET between the zinc porphyrin (ZnP) chromophore in zincsubstituted cytochrome c (Zn-cyt c) and an Alexa Fluor dye attached to specific surface sites was used to characterize Zn-cyt c unfolding. The use of ZnP as a fluorescent acceptor eliminates the need to doubly label the protein with exogenous dyes to perform FRET experiments in which both donor and acceptor fluorescence is monitored. The requirement for attachment of only one dye also minimizes perturbation to the protein. This sensitive technique allowed for the determination of distances between the label placed at six different sites and ZnP through a range of denaturant concentrations. Fitting of the data to a three-state model provides distances in the unfolding intermediate. The use of ZnP as a fluorescent acceptor of energy in FRET has a significant potential for application to a range of other systems including heme-binding proteins and proteins to which a covalently attached heme tag may be added.FRET ͉ protein folding ͉ heme F luorescence resonance energy transfer (FRET) is a simple and widely used technique for measuring molecular distances. FRET involves the nonradiative energy transfer between donor and acceptor fluorophores, the efficiency of which depends strongly on donor-acceptor separation (1). Structure, dynamics, and conformational changes of biomolecules including proteins, RNA, DNA, and polypeptides are amenable to study using FRET (2). As a biomolecular ''ruler,'' FRET is most useful when both donor quenching and acceptor enhancement of fluorescence are observed, because the presence of multiple nonradiative pathways for the donor can potentially cause inaccurate distance measurements if only donor f luorescence quenching is monitored.Because proteins are not readily synthesized chemically, preparing a pure sample of protein site-specifically labeled with two fluorophores appropriate for FRET is technically difficult and time-consuming. The most convenient and widely available chemically specific approach is to label Cys thiols, because Cys is an infrequently occurring amino acid. However, preparing a sample with the donor attached to one particular Cys and acceptor to a second particular Cys requires multiple purification steps, if it can be achieved at all (3, 4). Therefore, the majority of intramolecular FRET studies of proteins make use of an intrinsic fluorophore as one member of the donor-acceptor pair, with tryptophan the most commonly used (2). This approach allows two-color FRET studies on a sample labeled with only one attached extrinsic dye. Although tryptophan is advantageous in that it is naturally occurring and can be introduced (or removed) as needed through site-directed mutagenesis, it is easily quenched by surrounding amino acids, complicating data interpretation (5, 6). Additionally, tryptophan fluoresces in the UV, making it difficult to differentiate tryptophan fluorescence from other parasitic sources of fluorescence; a recent study highlighted the need to separate tryptophan fluorescence from intrinsic porphyri...

derived from propionic acid such as ibuprofen and naproxen should be avoided as they add an extra propionic acid load to the body.During maintenance of anesthesia, events such as hypoxemia, dehydration, hypotension, and acidosis should be avoided. During the fasting period, patients require IV fluids containing dextrose and sodium bicarbonate to suppress protein catabolism and subsequent acidosis. Supporting Romano et al (7), we also suggest that adequate protein restriction and carnitine administration should be maintained during peri-and early postoperative period as a precaution against metabolic decompensation and possible late complications.Clinical prognosis and long-term outcome depend essentially on the metabolic control to minimize accumulation of toxic metabolites. The findings in our 2 recipients are according to the most recent reports suggesting that LT has an important role in this setting, leading to clinical improvement, reflected by better feeding, fewer episodes of metabolic acidosis; hampering of neurocognitive decline, and improving cardiomyopathy (8).Accumulated cases from the literature also demonstrate that LT often obviates the need for dietary restriction and medical management. In fact, our cases achieved a clinical resolution of metabolic derangement and better quality of life with an average natural protein ingestion of 1 mg Á kg À1 Á day À1 . Serum propionylcarnitine levels, however, did not decrease markedly, mostly owing to the presence of extrahepatic sources of propionic acid (ie, the gut and the central nervous system). Thus, in addition to addressing the biochemical disorder, drastically reducing the incidence of hyperammonemia, functional cure is only partially complete making the role of LT not straightforward. In less severe cases, parents and physicians are balanced between conservative management and LT, the procedure in which access remains highly limited by donor shortage.

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