4 CONCLUSIONS

It is clear from the data that following rinsing, residual enzyme does remain in the Whatman paper in extremely small amounts, and how much seems to depend upon the purity (and concentration) of the enzyme: 0.5 μg of the pure amylase A6380/sq cm of paper versus 5 micrograms of the crude amylase A0273/sq cm of paper. Whether this residual enzyme is detrimental to the paper is not known and requires further experimentation. It would seem to be good practice, however, to remove as much of the enzyme and related materials as possible following conservation treatment. It is generally known that proteins tend to darken upon aging, and because enzymes are proteins, they may discolor if left behind in the paper.

It is also possible that residual enzymes could become reactivated sometime following conservation treatment. For example, a reactivated residual amylase might cause the reversal of a repair, hinge or lining if starch was the adhesive used. The conditions that might cause such a reversal have not been examined, but it seems expedient to recommend as thorough a removal of the enzyme as possible.

The data also suggest that the retention of enzyme by the paper appears to be an inherent property of the paper and is not necessarily caused by the presence of substrate on the paper. In addition, the amount of enzyme retained by the paper increased as the enzyme concentration increased. This finding suggests that the minimum amount of enzyme should be used when treating a paper artifact. Following this experiment, it was discovered that the enzyme A6380 can be used in extremely low concentrations, for example, 0.028% for an immersion treatment or 0.28% for local application (Baker, unpublished). Therefore the use of pure enzymes in such small concentrations followed by a few water rinses would tend to dispel many of the worries that paper conservators might have regarding the safe use of enzymes.

The data further suggest that sodium phosphate, not sodium acetate as mentioned in some conservation literature, is a more effective buffer for the tested enzymes both in the removal of substrate and the removal of the enzymes from the paper. At the present time, we can recommend, with some reservation, the use of a sodium phosphate buffer to buffer the enzyme solution or as a rinse. Any deleterious effect that this buffer may have on the aging properties of papers has yet to be determined. Nevertheless, if a porous paper is being treated, the use of a sodium phosphate buffer might be warranted to significantly reduce the amount of enzyme left behind.

While the results from these experiments using two amylases seem to indicate that simple water rinsing is efficacious, it is not known whether these data are generally applicable to the other common enzymes used in paper conservation such as protease and trypsin. Similar experiments using these enzymes should be performed to determine the most effective rinsing procedures. It is quite possible that in the case of the Whatman paper the gelatin surface-sizing helped to seal the paper from the amylase while the kizukishi did not have any such protection. If a protease is used on a protein surface-sized paper (or amylase on a starch surface-sized paper), the results might more closely resemble the ones obtained for the kizukishi. Presumably this result occurs because the enzyme can more easily get into an open paper structure but for some reason has a hard time getting out.

The differences in the results obtained for the Whatman and kizukishi papers may not depend wholly upon the existence of a surface size; they may be more closely related to the structure of these two very different papers. The Whatman paper is comprised of cotton fiber, probably chemically processed with a strong alkaline solution, perhaps bleached, mechanically beaten to yield relatively short fibers, and formed into a sheet that is quite compact and dense. The kizukishi, on the other hand, is made up of a bast fiber from the inner bark of a shrub. Chemical processing was probably limited to a mild wood ash cook with no chemical bleaching, hand beating, and formation into a thin, rather open sheet with the long fibers essentially intact. It is also possible that the neri (a natural mucilaginous formation aid, e.g. tororo-aoi) used in the formation of the Japanese paper may have some effect on the attraction and retention of the amylase in this experiment.

These are very interesting questions, and obviously further research is needed. But the conclusions that can be reached from this research can be summarized as follows:

1. Use the purest enzyme available. 6
2. Use as little enzyme as possible at its optimum pH.
3. Consider a prewater rinse with sodium phosphate if treating an already porous paper or a paper which will become more porous during the enzyme treatment.
4. Employ several rinses of room temperature water.

Judging from the results of these experiments, it does not seem advantageous to use a denaturant such as alcohol for a routine rinse. Although hot water, as either a rinse or a denaturant, was not tested, its use as a rinse would also seem to be redundant given the test results. It might still be advisable, however, to reserve these two techniques for an emergency, to stop an enzyme treatment immediately for example. Pretesting the paper and media with any of the solutions under consideration including emergency ones is, of course, highly recommended.

ACKNOWLEDGEMENTS

We gratefully acknowledge Marie Louise Hammarskj�ld and David Rekosh for allowing all the radioactive aspects of this investigation to be conducted in their laboratory facilities at the State University College of Buffalo, under the guidance of William W. Andrews. We also thank Robert Futernick for giving us the idea for this investigation and Debra Evans for her editing contributions. This research was carried out in spring 1990 by Theresa Meyer Andrews, under supervision of the other authors, as a partial requirement for the degree of master of arts and certificate of advanced study in art conservation. It was partially supported by a student award from the Faculty of Arts and Humanities, State University College at Buffalo.