Partial proteolytic protein maps: Cleveland revisited

ANALYTICAL

BlOCHEMISTRY

146, ~08-110

(1985)

Partial Proteolytic ANTHONY ~e~art~e~t

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Protein Maps: Cleveland

Revisited’

I. WALKER AND CARL W. ANDERSON~

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University,

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Received July 16, 1984 Low concentrations of sodium dodecyl sulfate have a dramatic effect on the partial proteolytic products obtained by digesting bovine serum albumin with chymotrypsin or trypin. The effect observed may be important far the inte~retation of peptide maps obtained by the method of D. W. Cleveland et ai. [( 1977) J. Biof. C&m. 252, 1102-l 1061. D 1985 Academic press inc. KEY WORDS: proteinase; Cleveland, sodium dodecyl sulfate; bovine serum albumin; chymotrypsin; peptides.

Since the description by Cleveland et al. in 1977 (l), acrylamide gel analysis of partial proteolytic digestion products has become one of the most commonly used techniques for comparing two proteins for sequence homology. A number of different proteinases including ~~u~~yl~coccu~ aureus V8, chymotrypsin, papain, subtilisin (l), and trypsin can be used to produce digestion products. The technique depends in part upon the fact that susceptible bonds in different contexts are cleaved at different rates; the existence of bonds that are cleaved at relatively slow rates gives rise to a pattern of partial digestion products characteristic of a given protein digested under a given set of conditions. Digestions are often performed in the presence of a low concentration of SDS3 using samples isolated from SDS-polyac~lamide gels. Usually the question being asked is: “Are two proteins from different sources identical or closely related?’ This would be

the conclusion if the patterns of partial digestion products were identical or very similar. The finding of a substantial difference between two patterns would suggest that the two proteins in question are unrelated or perhaps only remotely related. This latter conclusion is valid only if the two proteins have been prepared in comparable ways, a fact that too often is forgotten in the haste of obtaining a result. We recently obtained a result that graphically illustrates the importance of using comparable digestion conditions for comparisons of proteins. Figure 1 shows the partial proteolytic digestion products obtained from BSA digested with different amounts of chymotrypsin in bicarbonate buffer at pH 8. Similar results were obtained with trypsin. The digests a-g were performed in the presence of 0.05% SDS; the digests h-o were done in the absence of SDS. Without knowing that fact, the complete dissimilia~ty of the two patterns would lead most investigators to conclude that two unrelated proteins had been compared. The small amount of SDS that was necessary to completely alter the pattern obtained from the native protein could easily have resulted from carryover during sample isolation from an acrylamide gel. The effect of various concentrations of SDS on the proteolytic digestion of BSA was

’ The submitted manuscript has been authored under Contract DE-AC02-76CH00016 with the U. S. Department of Energy. The U. S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for gove~mental purposes, is acknowledged. ‘On sabbatical leave from the Biology Department, Brookhaven National Laboratory, Upton, N. Y. 11973. 3 Abbreviations used: SDS, sodium dodecyi sulfate; BSA, bovine serum albumin.

0003-2697/S

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Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

108

PARTIAL

PROTEOLYTIC

PROTEIN

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109

MINUS SDS

PLUS SDS -4 a b

MAPS

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CHYMOTRYPSIN

DIGESTION OF BSA

FIG. 1. Digestion of BSA by chymotrypsin in the presence or absence of SDS. BSA at 62 fig/ml was incubated with chymotrypsin (Worthington Biochemicals) in 0.05 M ammonium bicarbonate, pH 8, at 30°C for 20 min; digestions shown in (a) through (g) contained, in addition, 0.05% SDS. Each subsequent track (a-g, h-o) shows a digestion with half the chymotrypsin concentration used for the sample shown in the previous track: the chymotrypsin concentration was approximately 100 pg/ml in the samples shown in tracks (a) and (h). SDS-polyacrylamide gel electrophoresis was as described (I). Numbers at the leti of the figure indicate the position of protein size markers: /3-galactosidase, 116,000; phosphorylase a, 92.000; BSA, 68,000; glutamate dehydrogenase, 56,000; creatine kinase. 40,000: glyceraldehyde-3phosphate dehydrogenase, 35,000; carbonic anhydrase, 3 1,000.

examined further. At a BSA concentration of 62 &ml, a concentration of 0.02% SDS, the lowest concentration examined, was sufficient to alter the pattern of partial digestion products obtained with trypsin or chymotrypsin. Only relatively minor differences in band patterns were observed as the SDS concentration was raised to 0.25%; however, the SDS concentration had a significant effect on the extent to which the full-length molecule was digested and the rate at which partial proteolytic products were digested (data not shown). At low SDS concentrations, partial products appeared to be digested in preference to the full-length molecule. The rate of product digestion increased with in-

creasing SDS concentration up to 0.1%; at 0.25% SDS, the rate of digestion decreased. In contrast to results obtained with trypsin or chymotrypsin, the effect of SDS on the pattern of digestion products obtained with S. aureus proteinase V8 was much less dramatic (results not shown). The fact that protein tertiary structure affects the rate of proteolytic cleavage at particular sites is, of course, not new. We were, however, surprised by the magnitude of the effect in the case discussed above. Although we have only examined BSA, it is well known that proteins differ greatly in their susceptibility to SDS-induced denaturation, and we can conceive of circumstances

110

WALKER

AND ANDERSON

in which even experienced investigators might inadvertently obtain results as apparently misleading as those shown in Fig. 1. We have noted a lack of concern exhibited by some authors with regard to sample preparation for “Cleveland” maps. Therefore we offer this communication as a warning to those who might be less aware of the effect tertiary structure can have on proteolytic digestions. One method of lessening the likelihood of structure-induced artifacts would be to digest mixtures as well as the individual samples.

ACKNOWLEDGMENTS We thank R. J. Jackson and T. Hunt for providing space and facilities. These studies were supported by research grants from the Medical Research Council of Great Britain (to R.J.J.) and by the U. S. Department

of Ene,.sy

REFERENCE 1. Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli. U. K. (1977). J. Bid Chem. 252. 1102-I 106.