REFLEXIONES FINALES

3.2.1 Synthesis of N-hydroxysuccinimide monoester of hemin (hemin NHS-monoester).

In our hands, the published procedures for NHS esters of tetrapyrroles (Tamura et al., 1973; Roberts et al., 1987; Bedel-Cloutour et al., 1991; Obataya et al., 2000; Sakamoto et al., 2004) gave only moderate yields. To optimize the reaction conditions, two alternative reagents for the coupling [thionyl chloride and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC)], two solvents (DMF and CH2Cl2), as well as different reagent ratios and reaction times were tried (Table 3.2). We found experimentally that EDAC hydrolyzed in aqueous buffer with a half-life of approximately 2 h. Thus, thionyl chloride was used instead. The esterification reaction was monitored via TLC with a developing solution of 0.5/9.0/0.5 MeOH/CH2Cl2/pyridine for the final reaction mixture of hemin modification. The optimized conditions were hemin and thionyl chloride (molar ratio 1:200) allowed to react in DMF under argon in the dark at 65 oC for 2 h, followed by sequential addition of NHS solution (200 eq in CH2Cl2/DMF) and triethylamine (200 eq) at 4 oC. The entire mixture was reheated under argon in the dark at 65 oC for 19 h. The TLC plate showed that more than 50% of the hemin was converted into esters.

3.2.2 Trial purification of hemin-NHS reaction product.

To determine useful HPLC conditions, hemin and hemin dimethyl ester were separated via a high-performance liquid chromatography (HPLC) reverse phase column (ZORBAX Eclipse XDB-C18, 4.6 × 50 mm, observation at 410 nm). Solvent A [3:2 volume ratio of phosphate buffer (0.1 M, pH 3.5) in MeOH] and solvent B (MeOH) were used as eluents. With 30% B gradient to 100% B over 10 min, hemin was eluted at 9.3 min and hemin dimethyl ester at 10.9

min. These elution conditions were used to separate a reaction in which EDAC was used as the coupling agent. There were three product peaks, with elution times of 9.3 min for the unreacted hemin, 9.9 min (assigned the hemin NHS-monoester), and 11.5 min (assigned the hemin NHS- diester). The corresponding peak area ratio was 25:5:1. It was felt that the HPLC elution times of the hemin and the monoester were too close for preparative separation. It was also noted that the monoester would be a mixture of the 6- and 7-isomers, and the reaction of either of these (or the diester), followed by expected hydrolysis of the second ester during the protease cleavage protocol) would all finally give the desired covalently bound heme. Therefore, in subsequent studies, a hemin-thionyl chloride-NHS product (mixture of hemin, hemin monoester and diester) was used in protein reconstitution without further purification.

3.2.3 Reconstitution of HSA and apoMb.

The hemin-thionyl chloride-NHS product mixture (2 eq heme) was diluted to ~1.0 M by DMF and added to the solution of commercial HSA (50 M in pH 8.2, 20 mM aqueous

phosphate buffer, 1 eq) at rt in dark. The volume ratio of DMF to aqueous buffer was kept at 1:10. After gently stirring for 24 h, the protein mixture was dried under vacuum and treated via 2-butanone/acid method to remove hemin that was not covalently bound (Asakura, 1978).

An attempt was made to separate HSA and hemin-linked HSA on a reverse phase HPLC column (ZORBAX S300 C8, 4.6  150 mm, 3.5 m) with solvent A (0.1% HCOOH in water, v/v) and solvent B (0.1% HCOOH in MeCN, v/v). Various elution protocols were tried, but there was only one peak, with spectral analysis at 280 and 410 nm overlapping. Thus, this

column did not separate the mixture. This HSA reconstituted product was then used directly in digestion experiments without further purification.

ApoMb was prepared according to the literature (Asakura, 1978). The reconstitution of Mb was done using the same procedure as described above for HSA.

3.2.4 Glu-C digestion of the commercial and reconstituted HSA.

Standard digestion tests on commercial HSA were run under nonreducing nondenatured conditions (Kouzuma et al., 2002), reducing nondenatured conditions (Bruce et al., 1999), and reducing denatured conditions (Wa et al., 2006) similar to those described in the literature. To optimize the digestion process of HSA, treatment of commercial HSA with and without sodium dodecyl sulfate (SDS) denaturation, with and without dithiothreitol (DTT) and iodoacetamide (IA), and different Glu-C digestion time lengths were tried (Table 3.3). SDS unfolds proteins by disrupting non-covalent bonds. DTT breaks the –S–S– bond between cysteines. IA is used to bind covalently to the –SH side chain of cysteine so the protein cannot form –S–S– bonds. The digested product was monitored by SDS-PAGE. In the optimized protocol, all the reagents were dissolved in NH4HCO3 aqueous buffer (pH 7.9, 100 mM). HSA (800 g/ml, 500 l) was mixed with SDS solution (1% of w/v, 100 l) and incubated at 55 oC for 60 min. To this mixture DTT (50 mM, 100 l) was added and incubated at 55 oC for 60 min. IA (50 mM, 210 l) was added, and the mixture was incubated for another 60 min at rt in dark. This protein mixture was filtered via a filter device during centrifugation to remove small molecules in the system. The volume of the purified protein was adjusted to 500 l by NH4HCO3 buffer. The purified protein was mixed with Glu-C (16 g/ml, 500 l) and incubated at 37 oC for 16 h. The digestion was terminated by

adding 10 l concentrated HOAc. These digested protein fragments were frozen immediately at –80 o

C, lyophilized, and cleaned by C18 Ziptip for MALDI measurement.

Reconstituted HSA was digested by Glu-C using the same procedure as for commercial HSA.

3.2.5 UV-visible spectroscopy.

The UV-visible spectra of the reconstituted HSA and Mb products were recorded [Varian 50 Bio spectrophotometer, 1.5 mL quartz Supracil cuvettes (Spectracell) with 1 cm path lengths]. The yield of reconstituted Mb was predicted using the extinction coefficients of apoMb (15.47 mM-1cm-1) and holoMb (ε280/holoMb = 34.4 mM-1cm-1, ε408/holoMb = 188 mM-1cm-1) (Tamura et al., 1973; Pace et al., 1995).

3.2.6 Mass spectroscopy.

The digested commercial HSA, the reconstituted HSA product, and the digested reconstituted HSA were each subjected to matrix-assisted laser desorption/ionization mass spectrometry (MALDI) using an ABI 4800 MALDI TOF-TOF analyzer (MALDI TOF-TOF). The digested reconstituted HSA was also analyzed by HPLC/ESI-MS (AB SCIEX API 3200TM LC/MS/MS triple quadrupole mass spectrometer equipped with an orthogonal Turbo ion spray source and Agilent 1200 Series HPLC System). HPLC was run with 5% solvent B (0.1%