294s Biochemical Society Transactions ( 1 99 1 ) 19 Proteolysis of lung elastin by human neutrophil elastase SHARON HANNAH and ANDREW P. RYLE Department of Biochemi,stry, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, EH8 9XD, SCOTLAND. The enzymic destruction of the insoluble structural protein elastin by human neutrophil elastase (HNE) during a period of pr0teinase:antiproteinase imbalance has been implicated in the lung disease emph sema [ l ] . Immunological investigations [23 demonstrated the presence of elevated levels of peptides containing desmosine in the urine of atients suffering from chronic obstructive pu monary disease (COPD). However, in order to confirm such peptides were a result of HNE attack on lung elastin, it is necessary to isolate specific peptide products resultant from HNE digestion of lung elastin. The progression of the digestion ,of lung elastin by HNE was determined by measuring the soluble products of digestion as the production of free amino groups usin 2,4,6-trinitrobenzene sulphonic acid (TNBS) [$ This method does not measure amino groups t at remain insoluble. It has been adapted to allow the measurement, of total amino groups b using 2,6 -d ic h Io ro p he no I ind o p he n o I ( DC PI w h ic h is quantitatively reduced and decolourised by the sulphite by-product of the TNBS reaction. The insoluble peptide production m a then b e expressed as the difference between tKese values. Lung elastin digestion by HNE 1 : l o 0 w/w ratio of enzyme:substrate at pH7.4 and 7%) reached a maximum after 24 hours of digestion. Between 24 and 48 hours of di estion a decline in the amount of both the solu%le and insoluble termini was noted suggesting that some of the termini were modified making them unreactive to TNBS. This observation may be a result of coacervation 41; a process, attributed to the elastic, ydrophobic regions of the elastin molecule rather than the hydrophilic, desmosine cross-I in ked region. When the soluble peptide products of digestion were subjected to reversed phase chromatography using a gradient of acetonitrile they separated into 3 groups, the first bein the flow-throu h which contains the hydropKilic HNE. Tte remaining two groups were peptides derived from elastin. The earlier group maybe from the cross-link region and the late group from the elastic region of the molecule. Filtration experiments were performed in order to determine the size of the eptides. The late peptides were retained by a 10RD membrane. The proportion of material retained by the membrane increased between 24 and 48 hours, suggesting that between 24 and 48 hours an "aggregation" of the peptide material occurs to give a larger complex.





Abbreviations used: COPD, chronic obstructive pulmonary disease; DCPIP, 2,6-dic h lor0 he no I indo he no1; H N E, h u man neutrophil e astase; A B S , 2,4,6-trinitrobenzene sulphonic acid.


Immunological studies [5] have suggested that elastin contains two classes of epitopes which are reco nised by antisera to either the crosslinkec? region of elastin or the hydrophobic elastic region. Therefore, the eptides will be screened with a monoclonal anti ody recognising a hexapeptide unit of the elastic, hydrophobic region in an attempt to determine if the late peptide group is derived from this region of the elastin molecule. This in turn would add weight to the idea that the peptides from this region of the molecule are coacervating to produce a complex which is less responsive to TNBS. Further evidence will be produced by determining the amino acid composition of the peptide groups; high alanine levels are indicative of the cross-link re ion and high proline and valine indicative of w e hydrophobic region.


We acknowledge the Faculty of Medicine for providing a Studentship awarded to S.H. 1. Laurell, C.B. & Erikkson, S. (1963) Scand. J. Clin. & Lab. Invest. 15, 132-140. 2. Kucich, U., Christner, P., Li pman, M., Kimber, P., Williams, G., Rosenbloom, & Weinbaum, G. (1985) Am. Rev. Res ir Dis 131, 709-713. 3. Satake, K., Okuyama, Ohasi, M. & Shinoda, T. J. Biochem. (Tokyo) 47, 654-660. .W. (1978) Perspect. Biol. Med. 2 1, 265-295. 5. Mecham, R.P. & Lange, G. (1982) Biochem. 21, 669-673.



Proteolysis of lung elastin by human neutrophil elastase.

294s Biochemical Society Transactions ( 1 99 1 ) 19 Proteolysis of lung elastin by human neutrophil elastase SHARON HANNAH and ANDREW P. RYLE Departme...
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