We previously reported the design of a library of de novo proteins targeted to fold into 4-helix bundles. 1 The library was created using a "binary code" strategy in which the sequence locations of polar and nonpolar amino acids were specified explicitly, but the identities of these side chains were varied extensively. Combinatorial diversity was made possible by the organization of the genetic code: Positions designed to contain polar amino acids were encoded by the degenerate DNA codon NAN, which codes for Lys, His, Glu, Gln, Asp, or Asn. Positions designed to contain nonpolar amino acids were encoded by NTN, which codes for Met, Leu, Ile, Val, or Phe (N represents a mixture of DNA nucleotides). We subsequently reported that approximately half the sequences in our initial library 1 bind heme. 2 The enormous diversity of sequences within the binary code library presents an opportunity for the isolation of de novo heme-based enzymes. We now establish the catalytic potential of the binary code proteins by demonstrating that several of the de novo heme proteins function as peroxidases.Natural peroxidases such as horseradish peroxidase (HRP) catalyze the reduction of hydrogen peroxide or alkyl peroxides to water or alcohol, respectively. The peroxidase mechanism is described by the following steps: 3 E represents the ferric resting state of the heme enzyme. Compound I is an intermediate two oxidation states above the resting state, and compound II is one oxidation state above the resting state. AH 2 is the reducing agent. Two commonly used reducing agents are 2,2′,5,5′-tetramethyl-benzidine (TMB) and 2,2′-azino-di(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS). These reagents become colored upon oxidation, thereby allowing peroxidase activity to be monitored spectraphotometrically.To search for novel peroxidases in our library of heme proteins, we developed an activity screen that does not require purification of the de novo proteins from cellular contaminants. Samples were prepared by a rapid freeze/thaw protocol 4 shown previously to produce protein samples of sufficient quality for various studies including NMR spectroscopy 5a and H/D exchange kinetics. 5b Freeze/thaw samples from 37 binary code proteins 6 were screened by monitoring oxidation of TMB. The results (Figure 1) indicate that several of the de novo proteins display activity significantly above the controls.On the basis of this screen, we chose four proteins for detailed analysis. 9 Proteins were purified, reconstituted with heme, and analyzed for peroxidase activity using ABTS as the reducing agent. Experimental data for protein 86 are shown in Figure 2. Neither the apoprotein nor the heme alone 10 were active, thereby demonstrating the heme-protein complex is the active species.Maximal velocities (V max ) were determined by plotting the data according to the Michaelis-Menten model (Figure 3). Turnover numbers (k cat ) were calculated by dividing V max by the concentration of heme protein. More detailed kinetic constants (k 1 and k 3 ) were determine...

Objective: To evaluate the morphologic changes in the macula of subjects with repaired macula-off retinal detachment (RD) using high-resolution Fourier-domain optical coherence tomography (FD OCT) and to perform functional correlation in a subset of patients using microperimetry (MP-1).Design: Prospective observational case series. Participants: Seventeen eyes from 17 subjects who had undergone anatomically successful repair for macula-off, rhegmatogenous RD at least 3 months earlier and without visually significant maculopathy on funduscopy.Methods: FD OCT with axial and transverse resolution of 4.5 m and 10 to 15 m, respectively, was used to obtain rapid serial B-scans of the macula, which were compared with that from Stratus OCT. The FD OCT B-scans were used to create a 3-dimensional volume, from which en face C-scans were created. Among 11 patients, MP-1 was performed to correlate morphologic changes with visual function.Main Outcome Measures: Stratus OCT scans, FD OCT scans, and MP-1 data.Results: Stratus OCT and FD OCT images of the macula were obtained 3 to 30 months (mean 7 months) postoperatively in all eyes. Although Stratus OCT revealed photoreceptor disruption in 2 eyes (12%), FD OCT showed photoreceptor disruption in 13 eyes (76%). This difference was statistically significant (PϽ0.001, 2 ). Both imaging modalities revealed persistent subretinal fluid in 2 eyes (12%) and lamellar hole in 1 eye. Among 7 subjects who had reliable MP-1 data, areas of abnormal function corresponded to areas of photoreceptor layer disruptions or persistent subretinal fluid in 5 subjects (71%); one subject had normal FD OCT and MP-1.Conclusions: Photoreceptor disruption after macula-off RD repair is a common abnormality in the macula that is detected better with FD OCT than Stratus OCT. A good correlation between MP-1 abnormality and presence of photoreceptor disruption or subretinal fluid on FD OCT demonstrates that these anatomic abnormalities contribute to decreased visual function after successful repair. Financial Disclosure(s): The authors have no proprietary or commercial interest in any materials discussed in this article.

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We report three members of one family, a mother and two daughters aged 4 and 7 years, who developed visual loss from Leber hereditary optic neuropathy within a 19-month period. All three had been exposed to smoke from two large rubber tire fires within the previous 24 months, suggesting the possibility of an epigenetic triggering factor.