Influence of Prosthetic Groups on Protein Folding and Subunit Assembly

Yip, Y. K.; Waks, Marcel; Beychok, Sherman

doi:10.1016/s0021-9258(19)44619-x

Cited by 121 publications

(30 citation statements)

References 31 publications

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“…34,35 Furthermore, hematin dimerization and polymerization alter its absorbance spectra, which leads to inaccurate measurement of the free heme concentration. 36,37 This is also demonstrated by the varying extinction coefficients of hematin observed in the literature ranging from 50 mM −1 cm −1 at 390 nm 38 or 385 nm 39 to 58.4 mM −1 cm −1 at 385 nm. 40 It has also been shown that the hematin Soret band in neutral aqueous solution does not obey Beer's law.…”

supporting

confidence: 64%

“…Additionally, in studies that quantified apoHb via the 280 nm absorbance, an offset at the equimolar absorbance point and varying heme:apoHb binding ratios have been presented. 70,75,79,80 Vasudevan and McDonald also reported a blue spectral shift (from 425 to 420 nm) while performing their DCNh titration. 27 However, the data presented here and studies performed with incorporation of DCNh into myoglobin indicated this shift was temporary.…”

Section: ■ Results and Discussionmentioning

confidence: 90%

“…Therefore, inaccurate quantification, slow reaction time, and large heme increments could also explain other inconsistencies in previous studies. Additionally, in studies that quantified apoHb via the 280 nm absorbance, an offset at the equimolar absorbance point and varying heme:apoHb binding ratios have been presented. ,,, …”

Section: Resultsmentioning

confidence: 99%

“…Additionally, previous studies have used hematin or carboxyheme (CO-heme) as the apoHb probe molecule, which could result in inaccurate and irreproducible active apoHb quantification. For example, hematin aggregates in aqueous solution, which complicates its binding to apoHb. , Furthermore, hematin dimerization and polymerization alter its absorbance spectra, which leads to inaccurate measurement of the free heme concentration. , This is also demonstrated by the varying extinction coefficients of hematin observed in the literature ranging from 50 mM –1 cm –1 at 390 nm or 385 nm to 58.4 mM –1 cm –1 at 385 nm . It has also been shown that the hematin Soret band in neutral aqueous solution does not obey Beer’s law.…”

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confidence: 99%

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Quantification of Active Apohemoglobin Heme-Binding Sites via Dicyanohemin Incorporation

2017

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Apohemoglobin (apoHb) is produced by removing heme from hemoglobin (Hb). However, preparations of apoHb may contain damaged globins, which render total protein assays inaccurate for active apoHb quantification. Fortunately, apoHb heme-binding sites react with heme via the proximal histidine-F8 (His-F8) residue, which can be monitored spectrophotometrically. The bond between the His-F8 residue of apoHb and heme is vital for maintenance of fully functional and cooperative Hb. Additionally, most apoHb drug delivery applications facilitate hydrophobic drug incorporation inside the apoHb hydrophobic heme-binding pocket in which the His-F8 residue resides. This makes the His-F8 residue a proper target for apoHb activity quantification. In this work, dicyanohemin (DCNh), a stable monomeric porphyrin species, was used as a probe molecule to quantify active apoHb through monocyanohemin-His-F8 bond formation. ApoHb activity was quantified via the analysis of the 420 nm equilibrium absorbance of DCNh and apoHb mixtures. His-F8 saturation was determined by the presence of an inflection point from a plot of the 420 nm absorbance of a fixed concentration of apoHb against an increasing DCNh concentration. Various concentrations of a stock apoHb solution were tested to demonstrate the precision of the assay. The accuracy of the assay was assessed via spectral deconvolution, confirming His-F8 saturation at the inflection point. The effect of the heme-binding protein bovine serum albumin and precipitated apoHb on assay sensitivity was not significant. An analysis of the biophysical properties of reconstituted Hb confirmed heme-binding pocket activity. Taken together, this assay provides a simple and reliable method for determination of apoHb activity.

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supporting

confidence: 64%

Section: ■ Results and Discussionmentioning

confidence: 90%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Quantification of Active Apohemoglobin Heme-Binding Sites via Dicyanohemin Incorporation

2017

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“…The Hb was isolated from red blood cells according to established procedures 28 and stored as the CO adduct. Apo-Hb was prepared by the acidacetone method 29 and reconstitution with mesodeuterated protoheme to form Hb-d 4 was done as described previously. 24,28,30 The Hb and Hb-d 4 were dissociated into their subunits by reaction with PMB 31 and isolated as reported in the literature.…”

Section: Protein Preparationmentioning

confidence: 99%

Resonance Raman studies of heme structural differences in subunits of deoxy hemoglobin

et al. 2000

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Low frequency resonance Raman (RR) spectra are reported for deoxy hemoglobin (Hb), its isolated subunits, its analogue bearing methine-deuterated hemes in all four subunits (Hb-d 4 ), and the hybrids bearing the deuterated heme in only one type of subunit, which are [␣ d4 ␤ h4 ] 2 and [␣ h4 ␤ d4 ] 2 . Analyzed collectively, the spectra reveal subunit-specific modes that conclusively document subtle differences in structure for the heme prosthetic groups in the two types of subunits within the intact tetramer. Not surprisingly, the most significant spectral differences are observed in the ␥ 7 mode that has a major contribution from out of plane bending of the methine carbons, a distortion that is believed to relieve strain in the high-spin heme prosthetic groups. The results provide convincing evidence for the utility of selectively labeled hemoglobin hybrids in unraveling the separate subunit contributions to the RR spectra of Hb and its various derivatives and for thereby detecting slight structural differences in the subunits.

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Prediction of β‐sheets in immunoglobulin chains. Comparison of various methods and an expanded 20 × 20 table for evaluation of the effects of nearest‐neighbors on conformations of middle amino acids in proteins

Szu

Jernigan

et al. 1978

Biopolymers

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SynopsisThe extraordinarily large number of immunoglobulins renders them an intriguing class of molecules for attempts to predict their conformations. The predictive method applied, using a 20 X 20 table of the observed effects of nearest-neighboring amino acids on the conformation (@,* angles) of the middle residue in known proteins, indicates positions of tripeptides that tend to break a-helices or regular 0-sheets. This 20 X 20 table is derived from data on 19 proteins, as compared with the earlier version based on 12 proteins, and includes a separate listing of residues of 0-turns that have helical +,* values. Secondary conformations predicted by methods of Chou and Fasman, Lim and Burgess, Ponnuswamy, and Scheraga have also been compared; for all three methods, wrong predictions of residues in 0-sheet conformation exceed correct ones. Better predictions are obtained when there is agreement with two or three of the methods. If there is consistent overprediction of @-structure, as with the Chou and Fasman method, the use of the 0-sheet-breaking tripeptides can improve predictability somewhat.

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Influence of Prosthetic Groups on Protein Folding and Subunit Assembly

Cited by 121 publications

References 31 publications

Quantification of Active Apohemoglobin Heme-Binding Sites via Dicyanohemin Incorporation

Quantification of Active Apohemoglobin Heme-Binding Sites via Dicyanohemin Incorporation

Resonance Raman studies of heme structural differences in subunits of deoxy hemoglobin

Prediction of β‐sheets in immunoglobulin chains. Comparison of various methods and an expanded 20 × 20 table for evaluation of the effects of nearest‐neighbors on conformations of middle amino acids in proteins

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