Heat-induced Cleavage Research Articles

Photodimers of 9-Haloanthracenes as Initiators in Atom Transfer Radical Polymerization: Effect of the Bridgehead Halogen

Photodimers of 9-chloroanthracene, formed by a [4+4] cycloaddition reaction of 9-chloroanthracene, were used as initiators in the atom transfer radical polymerization of styrene and compared to results previously obtained using photodimers of 9-bromoanthracene as the initiator. Heat-induced cleavage of the photodimer coupled with slow initiation from the bridgehead radical have been used to account for the lack of control in the systems, and thus changing the halogen on the initiating photodimer could support or refute this model. Reactions performed using analogous procedures produced polymers with number average molecular weight (Mn) values significantly higher in the case of 9-chloroanthracene photodimer-initiated systems, with similar polydispersity index (PDI) values observed in trials catalyzed with CuCl or CuBr. Polymer products showed absorbance bands indicative of regenerated anthracene in all cases, consistent with heat-induced cleavage of the photodimer during the course of the polymerization. Kinetic plots demonstrated that the polymerizations initiated with photodimers of 9-chloroanthracene showed maximum Mn values were obtained after approximately 10% monomer conversion, with a decline in Mn as a function of monomer conversion after this point. The data support slower initiation in the case of 9-chloroanthracene photodimers, followed by heat-induced cleavage throughout the polymerization system.

Quality Control of Photosystem II: CLEAVAGE OF REACTION CENTER D1 PROTEIN IN SPINACH THYLAKOIDS BY FtsH PROTEASE UNDER MODERATE HEAT STRESS

When spinach thylakoids were subjected to moderate heat stress (40 degrees C for 30 min), oxygen evolution was inhibited, and cleavage of the reaction center-binding protein D1 of photosystem II took place, producing 23-kDa N-terminal fragments. The D1 cleavage was greatly facilitated by the addition of 0.15 mM ZnCl2 and 1 mM ATP and was completely inhibited by 1 mM EDTA, indicating the participation of an ATP-dependent metalloprotease(s) in the D1 cleavage. Herbicides 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, bromoxynil, and ioxynil, all of which bind to the Q(B) site, inhibited the D1 cleavage, suggesting that the DE-loop of the D1 protein is the heat-sensitive cleavage site. We solubilized the protease by treating the thylakoids with 2 M KSCN and detected a protease activity in the supernatant by gelatin activity gel electrophoresis in the 70-80-kDa region. The antibodies against tobacco FtsH and Arabidopsis FtsH2 reacted with a 70-80-kDa band of the KSCN-solubilized fraction, which suggests the presence of FtsH in the fraction. In accordance with this finding, we identified the homolog to Arabidopsis FtsH8 in the 70-80-kDa region by matrix-assisted laser desorption ionization time-of-flight mass analysis of the thylakoids. The KSCN-solubilized fraction was successively reconstituted with thylakoids to show heat-induced cleavage of the D1 protein and production of the D1 fragment. These results strongly suggest that an FtsH protease(s) is involved in the primary cleavage of the D1 protein under moderate heat stress.

Mechanistic investigation of 9-bromoanthracene photodimers as initiators in atom transfer radical polymerization

Mechanistic pathways accounting for the lack of control in polymerizations employing photodimers of 9-bromoanthracene as alkyl halide initiators in atom transfer radical polymerization (ATRP) reactions are presented. Converting the aryl bromide on the anthracene moiety into an alkyl bromide via a [4+4] cycloaddition reaction effectively generated the photodimer with two alkyl halide sites, which were investigated as potential initiating sites for the ATRP of styrene and n-butyl acrylate. Polymers synthesized using these photodimers as initiators possessed relatively broad polydispersity index (PDI) values and displayed a non-linear relationship between their number average molecular weights (Mn) and monomer consumption, consistent with slow initiation from the bridgehead alkyl halide. Reactions performed at 80°C in bulk or THF generated polystyrene with Mn values 3–5 times higher than calculated based on monomer-to-initiator ratios. UV–vis spectrometry of the products demonstrated absorbance bands indicative of polymer-bound anthracene, caused by thermal degradation of the photodimer during the polymerization. When the initiator was introduced last into the reaction mixture in an attempt to suppress photodimer cleavage prior to initiation, PDI values and Mn values were generally lowered with the resulting polymers showing similarly high anthracene content. Composition of polystyrene and poly(n-butyl acrylate) products was also studied as a function of reaction temperature, with decreased anthracene labeling observed at lower temperatures (40 and 60°C), further validating a model of heat-induced cleavage of the photodimer.

Protection of DNA by salts against thermodegradation at temperatures typical for hyperthermophiles.

The effect of physiological concentrations of KCl and MgCl2 on the chemical stability of double-stranded and single-stranded DNA has been studied at temperatures typical for hyperthermophiles. These tow salts protect both double and single-stranded DNA against heat-induced cleavage by inhibiting depurination. High KCl concentration also protect DNA cleavage at apurinic sites, while high MgCl2 concentrations stimulate this cleavage. It has been previously proposed that salt protects double-stranded DNA against depurination by stabilizing the double helix. However, the inhibition of the depurination of single-stranded DNA by KCl and MgCl2 indicates that this effect is more probably due to a direct interaction of salts with purine nucleotides. These results suggest that the number and nature of heat-induced DNA lesions which have to be repaired might be quite different from one hyperthermophile to another, depending on their intracellular salt concentration. High salt concentrations might be also useful to protect DNA in long polymerase chain reaction (PCR) experiments and for long-term preservation.

Nondisulfide Crosslinking and Chemical Cleavage of Tubulin Subunits: pH and Temperature Dependence

Tubulin is known to be extremely unstable. The denaturation process partially involves irreversible aggregation, mediated by disulfide crosslinking. In addition, tubulin is known to undergo chemical cleavage during boiling in sodium dodecyl sulfate (SDS), a process that generates small peptides that have been mistaken for low molecular weight MAPs. Similar peptide cleavage has now been observed during two-dimensional denaturing isoelectric focusing-SDS-polyacrylamide gel electrophoresis. This phenomenon has complicated interpretation of limited proteolysis studies of tubulin by subtilisin. In an effort to avoid this problem we have undertaken a detailed study of the solution conditions that promote chemical cleavage of tubulin. The cleavage reaction is found to be strongly pH, time, and temperature dependent. Nondenatured and denatured tubulin is susceptible to peptide cleavage, suggesting that primary structure is more important than secondary structure in selection of susceptible bonds. After transfer of cleavage products from an SDS gel to a polyvinylidene difluoride membrane, amino acid sequencing has confirmed cleavage at Asp-Pro bonds, at position 306 in α-tubulin and at position 304 in β-tubulin. We also infer cleavage at the only additional Asp-Pro peptide bond located at position 31 in β-tubulin. Heat-induced cleavage at Asp-Pro accounts for 5 of the 13 bands observed on SDS gels. In addition, a minor α-tubulin band has been sequenced from a two-dimensional gel, corresponds to cleavage at Asp-Cys located at α-tubulin position 200, and accounts for two additional bands observed on SDS gels. Under non-denaturing and nonpolymerizing conditions tubulin undergoes extensive intermolecular, disulfide crosslinking. At elevated temperatures and high pH, a small fraction of the crosslinking is not reduced by β-mercaptoethanol. Disulfide-crosslinked aggregates are not suspected because carboxymethylation of tubulin does not prevent their formation. Lysinoalanine has been found by amino acid analysis and thus covalent lysine-dehydroalanine crosslinks are suspected. Dehydroalanine is formed by β-elimination at serine and thus the presence of lysinoalanine is consistent with cleavage at Gly-Ser peptides, the most unstable serine peptide bond, and accounts for most of the remaining cleavage data.

Synthesis of N7-ethyldeoxyguanosine 5′-triphosphate and placement of N7-ethylguanine in a specific site in a synthetic oligodeoxyribonucleotide

N 7-Ethyldeoxyguanosine 5′-triphosphate ( N 7-EtdGRP) was synthesized by direct ethylation of dGTP with diethyl sulfate and purified by TLC on cellulose plates at ∼5% yield. N 7-EtdGTP was identified by its uv spectra at pH 1, 7.4, and 13, by its absorbance maxima and minima, and by the lability of the glycosidic bond to acid- and heat-induced cleavage. At pH 7.4, spontaneous cleavage of the glycosidic bond proceeded with a half-life of >48 h. An enzymatic method for placing an N 7-ethylguanine in a specific site in DNA was developed using terminal deoxynucleotidyltransferase and the 3′ to 5′ exonuclease and 5′ to 3′ polymerase of the Klenow fragment of Escherichia coli DNA polymerase I. The method should be readily adaptable to other modified bases as long as the modification does not occur at a base-pairing site (e.g., 5-methylcytosine, N 6-methyladenine, and others).

Heat-induced fragmentation of human alpha 2-macroglobulin.

Previous studies have demonstrated that human plasma alpha 2-macroglobulin (alpha 2 M) possesses a single subunit chain (Mr approximately 185,000) when incubated with dodecyl sulfate and dithiothreitol at 37 degrees C and analyzed by dodecyl sulfate-gel electrophoresis. The present study details the observation that heating alpha 2 M to 90 degrees C under identical conditions produces at least two additional polypeptide chains, termed bands II and III, with apparent molecular weights of 125,00 and 62,000. The generation of these fragments is enhanced by increasing the time of incubation. The appearance of band II composition of the buffer, dodecyl sulfate concentrations, or alpha 2 M protein concentration in the incubation mixture. The electrophoretic bands II and III of alpha 2 M have dissimilar 125I-labeled tryptic peptide digests and also differ in their amino acid composition. The heat-induced fragmentation of alpha 2M is not affected by the inclusion of a variety of low molecular weight protease inhibitors, suggesting that the appearance of bands II and III is not due to enzyme-catalyzed hydrolysis. When the subunit chain of alpha 2M is first cleaved by trypsin into the previously described Mr = 85,000 derivative, neither band II nor III material, nor other lower molecular weight products are generated by heat treatment. Furthermore, preincubation of alpha 2M with methylamine prevents fragmentation of the subunit chain. These results indicate that these fragments are neither pre-existing subunits of alpha 2M nor derivatives formed prior to treatment for gel analysis. These data provide evidence that a covalent bond in the alpha 2M molecule is unusually susceptible to heat-induced cleavage.