ABSTRACT A. 'Hypersensitive' peptide bonds and autodegradation of proteins Several pure proteins, which gave a single band on electrophoretic analysis, when stored for a long time, were found to be partially degraded. When the pure protein was incubated at 37 C in a buffer at physiological ph, for time periods ranging between 24 hours to 120 hours, a limit digest was obtained, the electrophoretic pattern of which was identical to that obtained with the stored protein. Degradation upon storage was not seen in all proteins; certain proteins when analysed even after a few years of storage showed no evidence of any degradation products. When such proteins were incubated at 37 C in the buffer at physiological ph no degradation was seen even after an extended period of time (120 hours). Such incubation was used as an assay method to identify proteins that were unstable. Among the twenty four c0tnmercially available proteins that were assayed, the unstable ones included alcohol dehydrogenase, penicillinase (S.aureus, B.cereus, E.cloacae), a-casein, ~ casein, actin (bovine, chick), ornithine decarboxylase and ribosomal s 4 and s 8 proteins. P lactoglobin, ~-galactosidase, carbonic anhydrase, ovalbumin, phosphorylase-b, lysozyme, BSA, crystallins (a, Pu and PL), aprotinin, cytochrome-c and soybean trypsin inhibitor were found to be stable. Since alcohol dehydrogenase (ADH) was found to be a very good substrate for autodegradation, extensive studies were done on this protein. The process of degradation was established to be a genuine autodegradation reaction by ruling out [1] the presence of trace amounts of proteases contaminating the commercial protein samples and [2] the presence of free radicals or metal ions in the assay buffer which could be a cause for the fragmentation observed in unstable proteins. Unstable proteins like ADH, penicillinase and ~-casein were found to undergo increased autodegradation upon increasing the temperature from 0 C to 56 C; stable proteins (~-crystalviii
lin, carbonic anhydrase and BSA) remained intact till 45 C or 56 C. The pattern of degraqation was found to be same at all time points showing that a definite set of labile sites were available for cleavage and that the process of cleavage was non-random. ADH was also fow1d to undergo increased degradation and denaturation upon increasing the time of incubation. Denaturation of ADH preceded autodegradation but was not found to be a pre-requisite for degradation.! Increasing the ph of the incubation buffer also increased the degradation of ADH. In ~ casein no linear relationship between extent of degradation and increase in ph was seen. Addition of metal ions like calcium and magnesium had no effect on the autodegradation of ADH, but addition of zinc was found to stabilise this protein. In another unstable protein, penicillinase, none of the metal ions (Ca.++, Mg++ or zn++) had any effect on autodegradation. In this case EDT A was found to inhibit the reaction. Removal of oxygen from the buffer caused a slight decrease in the formation of the degradation products. The unstable proteins like ADH, actin and penicillinase were found to be slightly more susceptible to cleavage by H 2 o 2 (added extraneously) than stable proteins like BSA and carbonic anhydrase. The cleavage patterns of w1stable proteins in the presence of H 2 o 2 was found to be exactly similar to the patterns got on autodegradation of these proteins. Carboxymethylated ADH underwent autodegradation giving a totally different pattern from that of native ADH on SDS-PAGE (after autodegradation), indicating a change in the sites exposed for cleavage following change in native conformation. In the presence of 8 M urea, native ADH, carboxymethylated ADH and penicillinase did not undergo any autodegradation, showing either that urea stabilised the protein structures and prevented degradation or that a certain minimum of native conformation was essential for autodegradation. ADH, P-casein and actin were found to be more susceptible to cleavage by proteolysis than carbonic anhydrase, BSA and u and P crystllilins. The limit digests got by the action of BSPL-protease-1 on ADH and penicillinase were very similar to their respective autodegraded patterns. A limit digest of ADH with a non-specific protease, papain, also gave a sintilar pattern i.x
indicating that the sites of cleavage in both cases (protease digest, autodegraded) showed a lot of overlap. This was confirmed by sequencing a few of the peptides obtained on the digestion of ADH with BSPL protease and papain. Some sites in ADH which were cleaved during BSPLprotease digestion were also cleaved during autodegradation of ADH. Similarly, some bonds cleaved by papain were also found to be cleaved during autodegradation. The above observations suggested that autodegradation of a protein is primarily dependent on factors intrinsic to the structure of the protein. Peptides obtained on autodegradation of ADH, penicillinase, ~-casein, actin and ODC were each isolated, sequenced and the sites involved in cleavage were identified. The results indicated a preponderance of gly and, to a smaller extent, glu, ala, val, leu and a few other ~o acids at the degradation sites. Although gly and ala were present at a much higher frequency in ADH itself and glu was seen to occur at a higher frequency in ~-casein, the possibility of the cleavages being random was ruled out by observations of consistent limit digest patterns under a variety of experimental conditions. Studies on the synthetic peptides of calmodulin, which showed great differences in their susceptibilities to autodegrade, also suggested the possible role of a labile peptide bond(s) in making some of these peptides unstable. All these results showed that sequence-specific factors like labile or 'hypersensitive' peptide bonds could play a role in determining the stability of a protein or peptide. The observations of, [i] an absence of autodegradation in the presence of urea, [ii] a changed degradation pattern after carboxymethylation of ADH and [iii] the similarity in the electrophoretic patterns of autodegraded and protease-catalysed limit digestions of ADH, showed that the conformation of peptide chains containing the cleavage sites determined which of the labile peptide bonds in the protein would undergo cleavage. Many of the cleavage sites identified in ADH were found to be clustered at certain regions of the protein sequence, and this could probably be explained from the above arguments. The localisation of most of the cleavage sites to the less ordered and more flexible regions in the 3-D structure of actin also confirmed this observation. X
To conclude, it can be stated that hypersensitive or intrinsically labile peptide bonds in peptide or protein sequences make them more susceptible to autodegradation or cleavage by non-specific proteases; whether or not cleavage occurs would depend on constraints imposed by the secondary and tertiary elements making up the conformation of the peptide or protein. B. Stability of a protease isolated from a psychrotroph A comparison of the stability characteristics of various proteins from an extremophile with homologous proteins from mesophilic sources might provide useful information on the detenninants of intrinsic stability of proteins. A study was undertaken to isolate and purify proteins from a psychrotrophic yeast, Candida humicola, to see if they show any unusual stability characteristics. This yeast was found to secrete an extracellular acid protease which was active at low temperatures. This protease was purified and compared with a similar protease from a mesophilic yeast (Candida albicans). The secretion of this protease into the culture medium was maximum when an unbuffered medium containing BSA as the sole nitrogen source was used and was low under conditions of nitrogen starvation. The secretion increased with an increase in the growth of the cells, reaching a plateau in the early stationary phase. The protease was purified on FPLC, where a major peak containing the protease (of mol. wt 36 kd) was successfully separated and found to be homogeneous on SDS-PAGE. This protease was active at temperatures ranging from 0 to 45 C, with optimum activity at 37 C. When hemoglobin and BSA were used as substrates, the enzyme showed maximum activity at ph 1 to 1.2. However. when casein was used as a substrate, the protease showed two peaks of maximum activity, one at ph 1.0 and the other between ph 5 and 7. The protease was completely inhibited by pepstatin and SDS and partially inhibited by TLCK and iodoaretamide. N~ metal ions were required for its activity. The enzyme was found to hydrolyse various synthetic substrates but was found to be more active on native proteins like BSA, casein, gelatin and melittin xi
and generated peptides that could be detected on a gel or HPLC, showing that it was an endopeptidase. The protease was thermolabile at temperatures above 37 C. The enzyme at 37 C was found to increasingly autodegrade on increasing the time of incubation and by 24 hrs of incubation no protease band was visible on the gel. The protease was resistant to at least twenty cycles of freeze-thaw and was stable over a wide range of ph values and salt concentrations. Although this acid protease from the Antarctic microorganism (C.humicola) has certain unique features which may be of commercial value, it was probably not the right protein of choice for studying protein stability. Since proteases are very well known to undergo autolysis by an intennolecular catalysis mechanism, it would have been almost impossible to distinguish the bonds cleaved intermolecularly from those cleaved intramolecularly. xii