BIOPOLYMERS VOL. 11, 1607-1612 (1972)

The Hydrolysis of Poly (L-Prolyl-Glycyl-L-Prolyl) by Bacterial Collagenase

ELVIN HARPER,* ARIEH BERGER, and EPHRAIM KATCHALSKI, Department of Biophysics, The Weizmann Institute of Science,

Rehovoth, Israel

Synopsis Poly (L-Prolyl-Glycyl-cProlyl), a polymer which resembles collagen by physical and

immunochemical criteria, has been shown to serve as a substrate for the highly specific bacterial collagenase obtained from Clostridium histolyticum. The postulated reaction products Gly Pro, Gly Pro Pro, and Pro Gly Pro Pro have been isolated. The enzyme has been employed as an analytical tool in elucidating the sequence of synthesized poly- mers of proline and glycine.

The collagenase from Clostridium histolyticum is an enzyme of high specificity which employs only collagen or its denatured product gelatin as a substrate. The specificity of the enzyme residues in its ability to cleave the bond between x and Gly in peptides of the nature of -Pro-x-Gly-Pro-y, liberating N terminal glycyl peptides.2 There is a strict requirement for the penultimate amino acid, particularly on the carboxyl side of the peptide bond.3

Previous studies have utilized random copolymers of glycine and pr01ine.~ Recent work by Engle et al.5 has resulted in the synthesis of a (Pro-Gly- Pro), polymer as a model for collagen. Enzymatic hydrolysis of this polymer would be further evidence for a resemblance to collagen. The known speci- ficity of the enzyme allows us to expect the following as products of the hydrolysis: Pro Gly Pro Pro, Gly Pro Pro, and Gly Pro. This paper pre- sents evidence for this postulated specificity.

MATERIALS AND METHODS

Collagenase A was prepared by the methods of Harper and Seifter.6 Pro Gly Pro polymers were prepared by the methods of Engel et al.5 (Pro Gly Gly), and (Gly Pro Ala), were gifts of Dr. W. Traub. The activity of the enzyme with polymers as substrate was determined according to the number of QI amino groups liberated as measured by the ninhydrin method

* Present address: The Developmental Biology Laboratory Massachusetts General Supported by N.I.H. Postdoctoral Fellowship

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Hospital, Boston, Massachusetts 021 14. 1F2 AM-34-222-01.

0 1972 by John Wiley & Sons, Inc.

1608 HARPER, BERGER, AND KATCHALSKI

of Rosen.' The reaction was also followed by the decrease in optical rota- tion as the enzymatic reaction proceeded using an ETGNPL automatic polarimeter type 143A optical and control unit, with a Honeywell recorder and meter set at the D line 589 mp. The temperature was kept constant in a water-jacketed cell holder. A 2-cm cell was employed. High-voltage paper electrophoresis was carried out according to Katz.8 Samples were applied 18 cm from the anode to Whatman #1 filter paper (105 cm X 46 cm). The buffer contained 23 ml of 90% formic acid, water to one liter, and approxi- mately 5 ml of 6N HC1 to bring the pH to 1.4. The paper was dried for 20 minutes at 60°C. Reaction products were detected by chlorination and treatment with o-tolidine reagent as follows: the paper was exposed to chlorine gas for 5 minutes and aerated for 15 minutes. Completeness of aeration was examined by testing a sample strip for reactivity to the o- tolidine reagent. The reagent solution was 2 gm o-tolidine (biphenyl 4,- 4'-deamino, 3,3'-dimethyl), 20 ml glacial acetic acid, 800 ml water, and 5 g potassium iodide. A positive reaction gave dark blue spots against a pale blue to white background which faded on standing. Alternatively, the paper was developed with ninhydrin solution containing 2.5 gm ninhy- drin, 25 ml water, 2 ml pyridine, and 500 ml of acetone and dried for 20 minutes at 60°C. Enzyme concentrations were determined by the method of Lowry et al.9 Amino acid chromatographic analysis was as described by Stein and Moore.'O Lithium bromide was obtained from British Drug Houses. Guanidine thiocyanate was a product of Eastman Kodak. Z Gly Pro Gly Gly Pro Ala was purchased from Mann Research Labora- tories, New York.

Electrophoresis was performed for 4 hours a t 3000 volts.

Synthesis of Gly Pro Pro

Benzyloxycarboxyl glycine chloride (0.5 g) dissolved in dry ether (25 ml) was added to Pro Pro HBr (0.5 g) in 1N NaOH (4 ml) with stirring at O"C, keeping the pH between 9 and 10 for 30 minutes with dropwise addition of 1N NaOH. The mixture was then stirred for 60 minutes at room tompera- ture. The ether was removed under vacuum and the pH of the water layer brought to 2 with 6N HC1, whereupon an oil precipitated. The oil was taken up in warm benzene. Petroleum ether was added to the solu- tion and an oil precipitated which crystallized upon standing under vacuum. The benzyloxycarbonyl group was removed with 33% HBr in glacial acetic acid and the product precipitated and washed with dry ether; yield 525 mg (97%). Amino acid analysis gave a 2: 1 ratio of proline to glycine. One spot was seen by chlorination after paper electrophoresis a t pH 1.4 or at pH 6.5 using Verona1 buffer.

Synthesis of Pro Gly Pro Pro

Benzyloxycarbonyl proline chloride (0.5 g) dissolved in dry ether was added to Gly Pro Pro HBr (0.6 g) in 1N NaOH (5 ml) with stirring for 30

COLLAGENASE 1609

minutes at 0°C. During this time the pH was kept between 9 and 10 with 1N NaOH. The mixture was then stirred for 30 minutes a t room tempera- ture. The ether layer was separated and the water layer made acid to pH 2 with 6N HC1 whereupon an oil precipitated which was dissolved in ethyl acetate. The solution was dried over N&S04 and the ethyl acetate evaporated which resulted in an oil. The oil was taken into solution with warm benzene. Petroleum ether was added and a precipitate developed. The precipitate was washed with petroleum ether. The benzyloxycarbonyl group was removed by addition of 33y0 HBr glacial acetic acid; yield 496 mg (80%). Amino acid analysis gave a ratio of proline to glycinc of 3 : l . One spot was detected on paper electrophoresis a t pH 1.4 and a t pH 6.5 in Verona1 buffer. The synthesis of Gly Pro was according to the method of Engel et al.5

CoIlagenase Cleavage of (Pro Gly Pro) in Polymer

To Pro Gly Pro polymer of average molecular weight 6000 (determined by the method of Yphantis") after fractionation on Sephadex G 505 at a concentration of 4.18 mg/ml in 0.05M maleate 0.001M calcium chloride pH 7.0 was added collagenase A to a final concentration of 6.7 y/ml. A portion of the solution was placed in the polarimeter cell a t 37°C. Ali- quots of 0.1 ml were taken from the solution in the bath at indicated time intervals and added to 0.1 ml of formic acid pH 1.4. At the completion of the reaction, samples were assayed by Rosen ninhydrin and by high- voltage paper electrophoresis a t pH 1.4. The ninhydrin values showed an increase which correlated with the decrease in optical rotation as seen in Table I and the appearance of reaction products by high-voltage electro-

TABLE I Cleavage of (Pro-Gly-Pro) Polymer by Collagenase

Ninhydrin increase, Polarimeter decrease, Time, minutes O.D. 560 mp microamps

3 21 60

100 200

39 90

120 154 180

40 148 318 415 574

phoresis. An aliquot of the reaction mixture from the polarimeter cell was streaked on Whatman #1 paper and run in a preparative manner with high-voltage electrophoresis. The positions of the reaction products were located by ninhydrin assay of sample and strips. The products were eluted with water. Complction of elution was measured by negative ninhydrin reaction of the eluted paper and by lack of reaction to chlorina- tion. From a Pro Gly Pro polymer of a molecular weight of 6000 one would expect to obtain 20 reaction products; 18 Gly Pro Pro peptides migrating as one spot, 1 Pro Gly Pro Pro peptide, and 1 Gly Pro peptide.

1610 HARPER, BERGER, AND KATCHALSKI

Intermediate peptides arise as the polymer is cleaved by the enzyme. As the reaction proceeds to completion the major peptides seen are those which travel the same distance as the markers. The Gly Pro Pro reaction product is the major peptide with the highest color yield as would be expected. No unexpected peptides were seen by paper electrophoresis nor was there any evidence for glycine or proline alone as further reaction products. Those products which ran at the same rate from the point of application as Pro Gly Pro Pro (40 cm), Gly Pro Pro (48 cm), and Gly Pro (58 cm) markers toward the cathode were submitted to amino acid analysis. The resulting ratio of proline to glycine in each case was as expected, 3 : 1 for Pro Gly Pro Pro, 2 : 1 for Gly Pro Pro, and 1 : 1 for Gly Pro. This is taken as proof that the reaction products are those one would obtain from the postulated scission of the polymer by the enzyme.

Effect of Lithium Bromide and Guanidine Thiocyanate on Collagenase

Engel et al.5 have shown that the (Pro Gly Pro), polymer exhibits a specific optical rotation dependence as the molarity of LiBr or guanidine thiocyanate (GSCN) is increased. This is considered evidence for the unfolding of the molecule. An examination of the effect of these reagents on the enzymatic rate of hydrolysis of the polymer was therefore attempted. However, the enzyme exhibited a pattern of inhibition at the relatively high concentration of reagents employed. Thus, GSCN at 0.1M completely inhibited the enzyme and LiBr a t 1.5M caused a 70% inhibition as mea- sured by the cleavage of the substrate Z Gly Pro Gly Gly Pro Ala which was monitored by Rosen ninhydrin increase and high-voltage paper electro- phoresis of the reaction products on Whatman #1 paper a t pH 3.5 for 40 minutes a t 3000 volts. The paper was developed with ninhydrin and another sheet treated by chlorination to view the Z Gly Pro Gly formed.

Specificity of Collagenase

Considering the known specificity of the enzyme, it should be possible to employ Clostridium histolyticum collagenase as an analytic tool in eluci- dating the sequence of synthesized polymers of proline and glycine. There- fore, 3.25 mg/ml (Pro Gly Gly), of molecular weight 3000, (Pro Gly Ala), 2200 molecular weight, 10.05 mg/ml, and (Gly Pro Ala), 6000 molecular weight 3.75 mg/ml were each dissolved in 0.05M maleate 0.001M CaClz pH 7.0 buffer and incubated with 50 r/ml of Collagenase A for 3 hours a t 37°C. The reaction was stopped by addition of formic acid buffer pH 1.4 and the products analyzed by the ninhydrin method of Rosen and high- voltage electrophoresis a t pH 1.4. If (Pro Gly Gly), was cleaved, the expected reaction products would be 1 Pro Gly peptide, 8 Gly Pro Gly peptides, and 1 Gly Pro Gly Gly peptide and these were found in keeping with the Pro fi: Gly Pro y specificity previously established. (Gly Pro Ala), was hydrolyzed to Gly Pro Ala and no other reaction product was found, as predicted by the specificity pattern of Gly Pro Ala-Gly Pro Ala-

COLLAGEN ASE 1611

Gly Pro Ala. no Pro-z-Gly-Pro-y sequence exists in this polymer.

(Pro Gly Ala), was not a substrate for Collagenase A since

DISCUSSION

Evidence has been presented to show that the (Pro Gly Pro), polymer serves as a substrate for the highly specific Clostridium histolyticum col- lagenase. The isolation of the expected reaction products and their characterization by synthesis and amino acid analysis confirms this view. The (Pro Gly Pro), polymer has been shown to resemble collagen by biophysical parameters by Engel et al.5 and also immunochemically by Borek and Sela.12s13 These workers have obtained cross reactions to ichthyocol (carp swim bladder collagen) antibodies with the synthetic polymer as antigen and alternatively to antibodies directed against the Pro Gly Pro polymer to ichthyocol as antigen. These data, coupled with the work presented here, tend to lend credence to the view that the (Pro Gly Pro), polymer is the synthetic compound which to this date most resembles collagen.

The use of the enzyme collagenase as a selective tool to elucidate the sequence of polymers of proline and glycine has been demonstrated. Treatment of the polymers (Pro-Gly-Gly), and (Gly Pro Ala), gave rise to the predicted products in each case. Thus the enzyme may be em- ployed to ascertain whether the sequence -Pro-x-Gly-Pro-y, where 2 and y are any amino acid, exists in a synthetic or native molecule. The number of such sequences can be calculated in random copolymers by the use of collagenase. Available bonds of this sequence mill give rise to only N terminal glycine peptides.

SUMMARY

The (Pro Gly Pro), polymer (SO00 mol wt) which resembles collagen by biophysical and immunochemical criteria has been shown to be a substrate for bacterial collagenase. The predicted reaction products have been isolated and characterized. Collagenase has been used as a selective tool to identify a specific region of the structure of polymers of proline, glycine, and alanine.

References 1. S. Seifter and P. M. Gallop, in The Proteins, Vol. 4, H. Neurath, Ed., Academic

2. S. Michaels, P. M. Gallop, S. Seifter, and E. Meilman, Biochem. Bwphys. Acta,

3. Y. Nagai and H. Noda, Bwchim. Biophys. Acta, 34,298 (1959). 4. J. Engel, J. Kurtz, E. Katchalski, and A. Berger, J. Mol. Biol., 17,255 (1966). 5. S. Seifter, P. M. Gallop, and C. Franzblau, Trans. N.Y. Acud. Sci., 23,540 (1961). 6. E. Harper and S. Seifter, J. Biol. Chem. (in press). 7. H. Rosen, Arch. Bwchem. Bwphys., 67,lO (1957). 8. A. M. Katz, W. J. Dreyer, and C. B. Anfinsen, J. Bwl. Chem., 234,2897 (1959).

Press, New York, 1966, p. 153.

29, 450 (1958).

1612 HARPER, BERGER, AND KATCHALSKI

9. 0. H. Lowry, N. J. Rosenbrough, A. L. Farr, and R. J. Randall, J . BWZ. Chem., 193,265 (1951).

10. W. H. Stein and S. Moore, J . BWZ. Chem., 211,915 (1954). 11. D. A. Yphantis, Ann. N.Y. Acad. Sci., 88,586 (1960). 12. F. Borek, J. Kurtz, and M. Sela, Bioehim. Biophys. Acta, 188,314 (1969). 13. M. Sela, B. Schechter, I. Schechter, and F. Borek, Cold Springs Harbor Symp.

Quant. BWZ., 32,537 (1967).

Received January 25,1972 Revised May 1,1972