Effects of bed compression on protein separation on gel filtration chromatography at bench and pilot scale.

BACKGROUND: Poorly packed chromatography columns are known to reduce drastically the column efficiency and produce broader peaks. Controlled bed compression has been suggested to be a useful approach for solving this problem. Here the relationship between column efficiency and resolution of protein separation are examined when preparative chromatography media were compressed using mechanical and hydrodynamic methods. Sepharose CL-6B, an agarose based size exclusion media was examined at bench and pilot scale. The asymmetry and height equivalent of a theoretical plate (HETP) was determined by using 2% v/v acetone, whereas the void volume and intraparticle porosity (ϵ p) were estimated by using blue dextran. A protein mixture of ovalbumin (chicken), bovine serum albumin (BSA) and γ'- globulin (bovine) with molecular weights of 44, 67 and 158 kDa, respectively, were used as a 'model' separation challenge. RESULTS: Mechanical compression achieved a reduction in plate height for the column with a concomitant improvement in asymmetry. Furthermore, the theoretical plate height decreased significantly with mechanical compression resulting in a 40% improvement in purity compared with uncompressed columns at the most extreme conditions of compression used. CONCLUSION: The results suggest that the mechanical bed compression of Sepharose CL-6B can be used to improve the resolution of protein separation. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Conformational mobility in the active site of a heme peroxidase.

Conformational mobility of the distal histidine residue has been implicated for several different heme peroxidase enzymes, but unambiguous structural evidence is not available. In this work, we present mechanistic, spectroscopic, and structural evidence for peroxide- and ligand-induced conformational mobility of the distal histidine residue (His-42) in a site-directed variant of ascorbate peroxidase (W41A). In this variant, His-42 binds "on" to the heme in the oxidized form, duplicating the active site structure of the cytochromes b but, in contrast to the cytochromes b, is able to swing "off" the iron during catalysis. This conformational flexibility between the on and off forms is fully reversible and is used as a means to overcome the inherently unreactive nature of the on form toward peroxide, so that essentially complete catalytic activity is maintained. Contrary to the widely adopted view of heme enzyme catalysis, these data indicate that strong coordination of the distal histidine to the heme iron does not automatically undermine catalytic activity. The data add a new dimension to our wider appreciation of structure/activity correlations in other heme enzymes.

Redox and spectroscopic properties of human indoleamine 2,3-dioxygenase and a His303Ala variant: implications for catalysis.

Indoleamine 2,3-dioxygenase is an important mammalian target that catalyses the oxidative cleavage of l-tryptophan to N-formylkynurenine. In this work, the redox properties of recombinant human indoleamine 2,3-dioxygenase (rhIDO) and its H303A variant have been examined for the first time and the spectroscopic and substrate-binding properties of rhIDO and H303A in the presence and absence of substrate are reported. The Fe(3+)/Fe(2+) reduction potential of H303A was found to be -30 +/- 4 mV; in the presence of l-Trp, this value increases to +16 +/- 3 mV. A variety of spectroscopies indicate that ferric rhIDO at pH 6.6 exists as a mixture of six-coordinate, high-spin, water-bound heme and a low-spin species that contains a second nitrogenous ligand; parallel experiments on H303A are consistent either with His303 as the sixth ligand or with His303 linked to a conformational change that affects this transition. There is an increase in the low-spin component at alkaline pH for rhIDO, but this is not due to hydroxide-bound heme. Substrate binding induces a conformational rearrangement and formation of low-spin, hydroxide-bound heme; analysis of the H303A variant indicates that His303 is not required for this conversion and is not essential for substrate binding. The Fe(3+)/Fe(2+) reduction potential of H303A variant is approximately 70 mV lower than that of rhIDO, leading to a destabilization of the ferrous-oxy complex, which is an obligate intermediate in the catalytic process. In comparison with the properties of other heme enzymes, the data can be used to build a more detailed picture of substrate binding and catalysis in indoleamine 2,3-dioxygenase. The wider implications of these results are discussed in the context of our current understanding of the catalytic mechanism of the enzyme.

Defining substrate specificity and catalytic mechanism in ascorbate peroxidase.

Haem peroxidases catalyse the H2O2-dependent oxidation of a variety of, usually organic, substrates. Mechanistically, these enzymes are very well characterized: they share a common catalytic cycle that involves formation of a two-electron oxidized intermediate (Compound I) followed by reduction of Compound I by substrate. The substrate specificity is more diverse, however. Most peroxidases oxidize small organic substrates, but there are prominent exceptions to this and the structural features that control substrate specificity remain poorly defined. APX (ascorbate peroxidase) catalyses the H2O2-dependent oxidation of L-ascorbate and has properties that place it at the interface between the class I (e.g. cytochrome c peroxidase) and classical class III (e.g. horseradish peroxidase) peroxidase enzymes. We present a unified analysis of the catalytic and substrate-binding properties of APX, including the crystal structure of the APX-ascorbate complex. Our results provide new rationalization of the unusual functional features of the related cytochrome c peroxidase enzyme, which has been a benchmark for peroxidase-mediated catalysis for more than 20 years.

Crystal structure of the ascorbate peroxidase-ascorbate complex.

Heme peroxidases catalyze the H2O2-dependent oxidation of a variety of substrates, most of which are organic. Mechanistically, these enzymes are well characterized: they share a common catalytic cycle that involves formation of a two-electron, oxidized Compound I intermediate followed by two single-electron reduction steps by substrate. The substrate specificity is more diverse--most peroxidases oxidize small organic substrates, but there are prominent exceptions--and there is a notable absence of structural information for a representative peroxidase-substrate complex. Thus, the features that control substrate specificity remain undefined. We present the structure of the complex of ascorbate peroxidase-ascorbate. The structure defines the ascorbate-binding interaction for the first time and provides new rationalization of the unusual functional features of the related cytochrome c peroxidase enzyme, which has been a benchmark for peroxidase catalysis for more than 20 years. A new mechanism for electron transfer is proposed that challenges existing views of substrate oxidation in other peroxidases.

Substrate binding and catalytic mechanism in ascorbate peroxidase: evidence for two ascorbate binding sites.

The catalytic mechanism of recombinant soybean cytosolic ascorbate peroxidase (rsAPX) and a derivative of rsAPX in which a cysteine residue (Cys32) located close to the substrate (L-ascorbic acid) binding site has been modified to preclude binding of ascorbate [Mandelman, D., Jamal, J., and Poulos, T. L. (1998) Biochemistry 37, 17610-17617] has been examined using pre-steady-state and steady-state kinetic techniques. Formation (k1 = 3.3 +/- 0.1 x 10(7) M(-1) s(-1)) of Compound I and reduction (k(2) = 5.2 +/- 0.3 x 10(6) M(-1) s(-1)) of Compound I by substrate are fast. Wavelength maxima for Compound I of rsAPX (lambda(max) (nm) = 409, 530, 569, 655) are consistent with a porphyrin pi-cation radical. Reduction of Compound II by L-ascorbate is rate-limiting: at low substrate concentration (0-500 microM), kinetic traces were monophasic but above approximately 500 microM were biphasic. Observed rate constants for the fast phase overlaid with observed rate constants extracted from the (monophasic) dependence observed below 500 microM and showed saturation kinetics; rate constants for the slow phase were linearly dependent on substrate concentration (k(3-slow)) = 3.1 +/- 0.1 x 10(3) M(-1) s(-1)). Kinetic transients for reduction of Compound II by L-ascorbic acid for Cys32-modified rsAPX are monophasic at all substrate concentrations, and the second-order rate constant (k(3) = 0.9 +/- 0.1 x 10(3) M(-1) s(-1)) is similar to that obtained from the slow phase of Compound II reduction for unmodified rsAPX. Steady-state oxidation of L-ascorbate by rsAPX showed a sigmoidal dependence on substrate concentration and data were satisfactorily rationalized using the Hill equation; oxidation of L-ascorbic acid by Cys32-modified rsAPX showed no evidence of sigmoidal behavior. The data are consistent with the presence of two kinetically competent binding sites for ascorbate in APX.

Role of histidine 42 in ascorbate peroxidase. Kinetic analysis of the H42A and H42E variants.

To examine the role of the distal His42 residue in the catalytic mechanism of pea cytosolic ascorbate peroxidase, two site-directed variants were prepared in which His42 was replaced with alanine (H42A) or glutamic acid (H42E). Electronic spectra of the ferric derivatives of H42A and H42E (pH 7.0, mu = 0.10 m, 25.0 degrees C) revealed wavelength maxima [lambda(max) (nm): 397, 509, approximately equal to 540(sh), 644 (H42A); 404, 516, approximately equal to 538(sh), 639 (H42E)] consistent with a predominantly five-co-ordinate high-spin iron. The specific activity of H42E for oxidation of L-ascorbate (8.2 +/- 0.3 U.mg(-1)) was approximately equal to 30-fold lower than that of the recombinant wild-type enzyme (rAPX); the H42A variant was essentially inactive but activity could be partially recovered by addition of exogenous imidazoles. The spectra of the Compound I intermediates of H42A [lambda(max) (nm) = 403, 534, 575(sh), 645] and H42E [lambda(max) (nm) = 404, 530, 573(sh), 654] were similar to those of rAPX. Pre-steady-state data for formation of Compound I for H42A and H42E were consistent with a mechanism involving accumulation of a transient enzyme intermediate (K(d)) followed by conversion of this intermediate into Compound I (k'(1)). Values for k'(1) and K(d) were, respectively, 4.3 +/- 0.2 s(-1) and 30 +/- 2.0 mM (H42A) and 28 +/- 1.0 s(-1) and 0.09 +/- 0.01 mM (H42E). Photodiode array experiments for H42A revealed wavelength maxima for this intermediate at 401 nm, 522 nm and 643 nm, consistent with the formation of a transient [H42A-H(2)O(2)] species. Rate constants for Compound I formation for H42A were independent of pH, but for rAPX and H42E were pH-dependent [pKa = 4.9 +/- 0.1 (rAPX) and pK(a) = 6.7 +/- 0.2 (H42E)]. The results provide: (a) evidence that His42 is critical for Compound I formation in APX; (b) confirmation that titration of His42 controls Compound I formation and an assignment of the pK(a) for this group; (c) mechanistic and spectroscopic evidence for an intermediate before Compound I formation; (d) evidence that a glutamic acid residue at position 42 can act as the acid-base catalyst in ascorbate peroxidase.