Combined solid‐phase and solution approach for the synthesis of large peptides or proteins
In the synthesis of large peptides or proteins, highly homogeneous segments are indispensable for a convergent strategy either on a solid‐phase resin or in solution. Employing Boc/Bzl chemistry to prepare fully protected segments with a free α‐carboxyl group from the solid support, base‐labile linkers are profitable for practical peptide synthesis since they require no special equipment. For this purpose, an N‐[9‐(hydroxymethyl)‐2‐fluorenyl]succinamic acid (HMFS) linker was adopted. Consequently, there must be high compatibility between the protecting groups of the segment and the anchoring group which is cleavable by treatment with morpholine or piperidine in DMF. Instead of using the 2‐bromobenzyloxycarbonyl (BrZ) group for the Tyr residue and the formyl (For) group for the Trp residue, both of which are the most susceptible protecting groups under these base‐catalysed conditions, the base‐resistant 3‐pentyl (Pen) and cyclohexyloxycarbonyl (Hoc) groups were introduced to the respective side‐chain functional groups. By applying the present strategy, the authors were able to rapidly synthesize homogeneous protected segments for use in the subsequent segment coupling in solution. In the present paper, the utility of the combined solid‐phase and solution approach is demonstrated by synthesizing muscarinic toxin 1 (MTX1) which binds to the muscarinic acetylcholine receptors. Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd.
Solution structure of a green mamba toxin that activates muscarinic acetylcholine receptors, as studied by nuclear magnetic resonance and molecular modeling.
The three-dimensional solution structure of the MTX2 toxin (65 amino acids and 4 disulfides) from the green mamba venom (Dendroaspis angusticeps), a toxin that activates the pharmacological M1 muscarinic acetylcholine receptors, has been determined by nuclear magnetic resonance and molecular modeling. Seventeen structures were calculated from 810 distance and 68 dihedral angle restraints using DIANA and X-PLOR. The average rms deviation between the 17 refined structures and the energy-minimized average structure is 0.95 A for the backbone atoms. The overall folding of MTX2 consists of three loops stabilized by the four disulfides and forming a two- and a three-stranded beta-sheet. This structure appears to be very similar to that of other snake toxins, such as neurotoxins, fasciculins, and cardiotoxins, that also possess the same three-finger fold. For instance, the RMSd for the backbone atoms between MTX2 and the curaremimetic toxin alpha (from Naja nigricollis), the acetylcholinesterase inhibitor fasciculin 1 (from Dendroaspis angusticeps), and the cardiotoxic toxin gamma (from Naja nigricollis) are 1.86, 1.87, and 2.04 A, respectively. Local differences are observed between this toxin and the other structurally related toxins. Some of these differences could be relevant for the functional specificity of MTX2. In particular, this toxin presents a large twist at the tip of loop II due to a bulge (V31, T32; N35) that accommodates an inserted amino acid in the loop. This spatial arrangement brings the side chain of K34 in the beta-turn of the loop to be aligned with the beta-sheet. Hypotheses about a possible functional role of this lysine are described. Other characteristics in the side-chain distribution that could be related to the MTX2 function are presented.
A device for uranium series leaching from glass fiber in HEPA filter
For the disposal of a high efficiency particulate air (HEPA) glass filter into the environment, the glass fiber should be leached to lower its radioactive concentration to the clearance level. To derive an optimum method for the removal of uranium series from a HEPA glass fiber, five methods were applied in this study. That is, chemical leaching by a 4.0 M HNO3–0.1 M Ce(IV) solution, chemical leaching by a 5 wt% NaOH solution, chemical leaching by a 0.5 M H2O2–1.0 M Na2CO3 solution, chemical consecutive chemical leaching by a 4.0 M HNO3 solution, and repeated chemical leaching by a 4.0 M HNO3 solution were used to remove the uranium series. The residual radioactivity concentrations of 238U, 235U, 226Ra, and 234Th in glass after leaching for 5 h by the 4.0 M HNO3–0.1 M Ce(IV) solution were 2.1, 0.3, 1.1, and 1.2 Bq/g. The residual radioactivity concentrations of 238U, 235U, 226Ra, and 234Th in glass after leaching for 36 h by 4.0 M HNO3–0.1 M Ce(IV) solution were 76.9, 3.4, 63.7, and 71.9 Bq/g. The residual radioactivity concentrations of 238U, 235U, 226Ra, and 234Th in glass after leaching for 8 h by a 0.5 M H2O2–1.0 M Na2CO3 solution were 8.9, 0.0, 1.91, and 6.4 Bq/g. The residual radioactivity concentrations of 238U, 235U, 226Ra, and 234Th in glass after consecutive leaching for 8 h by the 4.0 M HNO3 solution were 2.08, 0.12, 1.55, and 2.0 Bq/g. The residual radioactivity concentrations of 238U, 235U, 226Ra, and 234Th in glass after three repetitions of leaching for 3 h by the 4.0 M HNO3 solution were 0.02, 0.02, 0.29, and 0.26 Bq/g. Meanwhile, the removal efficiencies of 238U, 235U, 226Ra, and 234Th from the waste solution after its precipitation–filtration treatment with NaOH and alum for reuse of the 4.0 M HNO3 waste solution were 100, 100, 93.3, and 100%.