Studies on barley trypsin inhibitor. II. Structural changes induced by denaturants and their reversibility.

/ . Biochem., 79, 321-328 (1976)

Studies on Barley Trypsin Inhibitor

Taro OGISO, Masahide AOYAMA, Mihoko WATANABE, and Yoshio KATO Gifu College of Pharmacy, Mitahora 492-36, Gifu, Gifu 502 Received for publication, August 6, 1975

No change in the activity of a trypsin inhibitor from barley was observed on treatment with heat and denaturants such as urea and guanidine hydrochloride. Thus, the conformational properties of the inhibitor were investigated. CD spectra of the native inhibitor were analysed by a curve-fitting technique using the data for polyL-lysine. The results indicate that the inhibitor contains 41% a-helix, 25% iS-structure, and 34% unordered conformation. In the presence of 2—8 M urea or 3 M guanidine hydrochloride, the CD spectra were almost unchanged. On treatment at 100° for 10 min in 8 M urea the depth of the trough at 200—240 nm decreased considerably, while both 6 M guanidine hydrochloride and 6 M guanidine hydrochloride at 100° reduced the molar ellipticity of the negative band at 222 nm by about two-thirds. When inhibitor treated in 6 M guanidine hydrochloride at 100° was diluted 10- or 30-fold, the conformation was significantly reformed. The CD spectra at alkaline pH showed that the inhibitor also has resistance to alkali. As a result of difference spectrum studies, it was shown that the inhibitor had peaks at 294 and 285 nm and troughs at 290 and 280 nm, due to denaturation, in strong denaturing media, 8 M urea at 100° and 6 M guanidine hydrochloride. The positive and negative peaks, however, immediately disappeared on removal of the denaturant. From spectrophotometric titrations, the phenolic hydroxyl groups were found to be ionized above pH 10.5. Four tyrosine residues are rapidly titrated and the last one was ionized above pH 12.5 with partial inactivation. Cleavage of the disulfide bridges in the inhibitor induced a very marked decrease in the value of [0]ui, leading to complete loss of the inhibitory activity. A possible interpretation of these data is that the inhibitor contains a rigid disulfide loop, and the disulfide bridges contribute to the structural stability and reversibility of the conformational changes of the protein.

Some natural inhibitors are known to be stable to denaturants ( 1 ) and heat (2-); basic pancreatic trypsin inhibitor remains unaltered at 77° and pH 2.1 or in 6 M guanidine hydroAbbreviation : inhibitor.

chloride, and selective cleavage of the disulfide bridge Cysu-Cys»g considerably reduces the stability of the inhibitor to heat or guanidine hydrochloride ( / ) . Our previous communication reported the purification and some properties of Nijyo barley trypsin inhibitor (3). During the study the inhibitor was found to

RCM-inhibitor, carboxymethylated

Vol. 79, No. 2, 1976

321

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II. Structural Changes Induced by Denaturants and Their Reversibility

322

T. OGISO, M. AOYAMA, M. WATANABE, and Y. KATO

rotation [m], respectively, and were not corrected for the refractive index dispersion of the solvent. The mean residue weight of the inhibitor was assumed to be 110.9, as previously reported (3). The CD intensity wascalibrated with an aqueous solution of d-10camphorsulfonic acid (Jtn = 2.2 M"'-cm"' at 190 nm) (6~). All the measurements were made at room temperature (20-25°). The amount of a-structure was calculated from the CD spectra (7, 8), fH =

40,000

Difference Spectra—Difference spectra were measured with a Shimadzu MPS-50L spectrophotometer using the full scale to 0.1 absorbance unit, using tandem cells of 0.5 cm optical path. Spectrophotometric Titrations of Tyrosyl ResMATERIALS AND METHODS idues—For spectrophotometric titrations of the Materials—A barley trypsin inhibitor was tyrosyl residues in the inhibitor, the procedure purified from a single lot of barley, Hordeum of Sherman and Kassell was used ( ° ) . The distichum L. var. emend Lamark (Nijyo, Kanto- titrations were done using a Shimadzu MPSnakate) by the method described in a previous 50L spectrophotometer and a Metrohm Potenpaper (3). Bovine pancreatic trypsin [EC tiograph E 436. The inhibitor was dissolved 3. 4. 21. 4] (twice crystallized) was obtained from in 50 mM sodium phosphate buffer (pH 7.0) and Sigma Chemical Co. (St. Louis, U.S.A.). Urea titrated in a cuvette with 1.5 N NaOH. was recrystallized from 70 per cent aqueous Cleavage of Disulfide Bridges and Carboxyethanol and guanidine hydrochloride of special methylation of Sulfhydryl Groups—Reduction grade was used without further purification. of the disulfide bonds with mercaptoethanol Determination of Protein Concentration- and carboxymethylation of the sulfhydryl Protein concentrations were determined spec- groups with iodoacetic acid were carried out trophotometrically by measuring the absorb- by the method of Crestfield et al. (10), except ance (E{®m = 12.6) (5) at 280 nm with a Hita- that 6 M guanidine hydrochloride was used inchi 101 spectrophotometer. stead of 8 M urea as a denaturant. Assays of Inhibitory Activities—Assays for Amino Acid Analysis — Protein samples inhibitory activity were done by tryptic hy- were hydrolyzed at 105° with constant-boiling drolysis of casein, using the Folin-Ciocalteau hydrochloric acid in evacuated, sealed tubes method ( 4), as described in the previous paper for 24 hr. Analyses were performed using a (5). Nihon Denshi amino acid analyzer, model JLCCD and ORD Measurements—CD and ORD 3BC. measurements were carried out in a 0.1 mm path length cell at protein concentrations of RESULTS 0.75—1.0 mg per ml of 10 mM phosphate buffer (pH 7.0, except for the measurements at alEffect of Denaturants on Inhibitory Ackaline pH), using a JASCO Model ORD/UV-5 tivity—As shown in Table I, the inhibitory acrecorder equipped with a CD attachment. tivity was not affected by any of the denaturThe CD and ORD data were expressed in ants tested. No further changes were observed terms of molar ellipticity [8] and mean residue on standing for 15 hr in 8 M urea or 6 M gua/ . Biochem.


be quite heat-stable and resistant to denaturants (3). However, it was not possible to determine whether the thermal stability and resistance to denaturants are the result of a unique molecular organization or whether they are due to the presence of a special peptide loop in the inhibitor. In an attempt to obtain information bearing on this point, a study of the effects of some denaturants, such as urea, guanidine hydrochloride, heat and alkali, on the trypsin inhibitor has been initiated. This report deals with conformational changes in the inhibitor in the presence of denaturants and their reversibility as studied by means of CD and ORD measurements, difference spectra, spectrophotometric titration of the tyrosyl groups and cleavage of the disulfide bridges in the inhibitor.

REVERSIBLE STRUCTURAL CHANGES OF BARLEY TRYPSIN INHIBITOR

Conditions

Perturbants None

Inhibitory activity

—

ICO

2M

101

4M

107

8M

104

8 M at 80° for lOmin

100


108

Urea

Guanidine hydrochloride

3M

104

6M

111


102


107

nidine hydrochloride. Surprisingly, even after heat treatment at 100° for 10 min in 8 M urea or 6 M guanidine hydrochloride, conditions under which most proteins are completely denatured, the inhibitor showed unaltered inhibitory activity. The effect of alkaline pH on the

200

220

240

WAVELENGTH (nm)

TABLE II. Changes in the inhibitory activity of the inhibitor in alkaline media. To 2 ml of 0.1 M glycine buffer at the indicated pH, 0.5 ml of the inhibitor solution (2 mg/ml) was added and the mixture was allowed to stand for the indicated time at room temperature. 0.1 ml of the mixture was mixed with 2 ml of 0.4 M Tris-HCl buffer (pH 8.0) and the inhibitory activity was assayed. Inhibitory activity (%) pH 8.5 10.0 11.0 11.5 12.0 12.5 12.9

After 1 hr

After 22 hr

100 108 113 111 112 94 37

100 104 104 103 104 64 5

inhibitory activity is shown in Table II. The inhibitor was stable over a wide pH range up to pH 12.0; above pH 12.5 the inhibitor was partially inactivated and the extent of inactivation increased progressively on prolonged treatment above pH 12.5. CD and ORD Spectra in the Far-ultraviolet Region—The CD spectrum of the native inhibitor is shown in Fig. 1 by a solid line. The CD spectrum of the native inhibitor exhibits a negative band at 208 nm with a shoulder at

200

220

240

260

WAVELENGTH (nm)

Fig. 1. Far-ultraviolet CD and far-ultraviolet ORD spectra of the inhibitor. Solid curves, experimental results; closed circles, simulated values for 41% a-helix, 25% ^-structure, and 34% unordered structure, calculated from poly-L-lysine spectra in water. A, CD spectrum ; B, ORD spectrum, Bars indicate deviations.

Vol. 79, No. 2, 1976


TABLE I. Effect of urea and guanidine hydrochloride on the inhibitory activity of the inhibitor. Inhibitor (15 mg/ml) was dissolved in 0.1 M phosphate buffer (pH 7.0) and treated with perturbants at the indicated conditions at 20° for 1 hr, unless otherwise stated. 0.2 ml of the mixture was diluted with 15 ml of water and inhibitory activity was assayed.

323


324

200

220 240 WAVELENGTH (nm)

Fig. 2. Effect of urea on the far-ultraviolet CD spectra of the inhibitor. I, native inhibitor; II, in 4 M ; III, in 8 M ; IV, in 8 M urea at 100° for 10 min ; V, 10-fold dilution of IV.

200

220

240

WAVELENGTH (nm)

Fig. 3. Effect of guanidine hydrochloride on the far-ultraviolet CD spectra of the inhibitor. I, native inhibitor ^11, in 3 M ; III, in 6 M ; IV, in 6 M guanidine HCI at 100° for 10 min; V, 10-fold dilution of IV. / . Biochem.


222 nm, which are indicative of helical poly- Fig. 2 represents the CD spectrum of inhibitor peptide conformation. A semigraphical meth- heated at 100° for 10 min in 8M urea. The od of CD data analysis proposed by Greenfield depth of the trough at 200—240 nm was reand Fasman ( 1 1 ) was applied to the figure and duced to a considerable extent by this treatthe fitted values calculated from poly-L-lysine ment, suggesting that the secondary and terspectra were plotted as shown in Fig. 1. Sim- tiary structures of the inhibitor were partially ulated plots at wavelengths from 220 to 250 unfolded, but about two-thirds of the ordered nm gave 41% a-helix, 25% ^-structure, and structure remained unaltered. The [d]m of 34% unordered structure for the inhibitor. A inhibitor heated at 100° in 8 M urea was —9,015, good fit in the region of 220—240 nm was ob- indicating 22.5% a-helix. Thus, the inhibitor tained, though deviation between the calcu- has excellent resistance to the denaturant. The lated and observed spectra was found below CD spectrum of the heated inhibitor after 10218 nm. The discrepancy below 218 nm is fold dilution with water was found to be only probably due to both disulfide bridge and side slightly changed in comparison with the data chain effects. The magnitude of [6] at 222 nm before dilution, and 30-fold dilution of the inwas found to be —16,455. The helical content hibitor treated with heat and urea gave almost of native inhibitor was estimated to be 41.1 the same result. per cent, if a value of [d]tu of —40,000 is asThe effect of guanidine hydrochloride on sumed for completely helical polypeptide (7, the CD spectrum of the inhibitor is shown in 8). The ORD curve of the inhibitor is also Fig. 3. Three M guanidine hydrochloride inshown in Fig. 1. This shows that there is a duced no detectable change in the protein conlarge amount of helical structure in the pro- formation, while the spectrum in 6 M guanidine tein. hydrochloride was markedly changed, the depth In order to clarify the mechanism of the of the trough at 200-240 nm being significantsurprising resistance of the inhihitor to dena- ly decreased and the value of [0]ta falling to turants, the CD spectra of the inhibitor were one-third of the native one. Part of the sectaken under various conditions. The effect of ondary and tertiary structures, however, reurea on the CD spectrum is shown in Fig. 2. mained unchanged, as shown in Fig. 3. The It was found that in the presence of 2—8 M depth of the trough at 200-240 nm of inhibiurea the CD spectra in the 200—250 nm region tor heated at 100° for 10 min in 6 M guanidine remained almost unchanged. Curve IV in hydrochloride was also found to decrease fur-


TABLE HI. The values of [0] m and a-helical composition of the inhibitor and RCM-inhibitor in the presence or absence of denaturants. a-Helix

Inhibitor Native in 2 M urea in 4 M urea in 8 M urea in 8 M urea at 100° for 10 min Diluted (8 M hot urea) 10-fold» Diluted (8 M hot urea) 30-fold' in 3 M guanidine in 6 M guanidine in 6 M guanidine at 100° for 10 min Diluted (6 M hot guanidine) 10-foldE Diluted (6 M hot guanidine) 30-fold» in water at 100° for 10 min RCM-inhibitor RCM-inhibitor in 6 M guanidine

-16,455 -16,440 -16,450 -16,485

41.1 41.1 41.1 41.2

-9,015

22.5

-9,680

24.2

-9,735

24.3

-15,950 -5,414

39.9 13.5

-4,080

10 2

-11,620

29.1

-11,750

29.4

-16,440

41.1

-5,690

14.2

-750

1.9

200

* diluted 10- or 30-fold with water after treatment with 8 M urea or 6 M guanidine hydrochloride at 100° for 10 min. Vol. 79, No. 2, 1976

ditions and a large part, but not all, of the unordered structure produced by denaturants was reformed into ordered structure immediately after dilution or removal of the denaturants. The CD spectra at alkaline pH are shown in Fig. 4. The inhibitor was also found to have resistance to alkaline pH, and even in 0.1 N NaOH the inhibitor retained a considerable amount of ordered structure. Difference Spectra—As shown in Fig. 5, the difference spectra of the inhibitor in 2, 4, or 8 M urea vs. native inhibitor in 10 mM phosphate buffer (pH 7.0) had peaks at 285 and 294 nm, probably due to the solvent perturbation of tyrosine and tryptophan. The peaks, however, were present even in a strong denaturing medium, namely 8 M urea at 100°, although this medium caused the appearance of troughs at 290 and 280 nm. The troughs at 290 and 280 nm may be due to denaturation blue shift caused by the exposure of a few tryptophan and tyrosine residues buried inside the molecule. This suggests that the denaturation of the inhibitor with hot 8 M urea was not marked and that the inhibitor is extremely resistant to denaturation with urea. Tenfold dilution of the inhibitor treated with 8 M

220

240

WAVELENGTH (nm)

Fig. 4. Effect of pH on the far-ultraviolet CD spectra of the inhibitor. I, native inhibitor; II, at pH 11.0; III, at pH 12.0; IV, at pH 12.9; V, in 0.1 N NaOH. The measurements were carried out 20 min after preparation of the inhibitor solution. The buffer was 100 mM glycine-100 mM NaOH (pH 10.0-12.9).


ther and the a-helix content fell from 41.1% for native inhibitor to 10.2fo. The CD spectram of inhibitor treated with 6 M guanidine hydrochloride at 100°, after 10-fold dilution with water, approached that of the native inhibitor at 200-240 nm. This suggests that reformation of most of the helical and ^-structure occurs just after dilution or removal of the denaturant. After 30-fold dilution of the denaturant, the value of [6]Itt was almost the same as that obtained on 10-fold dilution. The CD spectrum of inhibitor treated with heat alone (100° for 10 min) was almost the same as the native one, as shown in Table III. These results indicate that part of the ordered structure of the inhibitor remained in spite of the extremely severe denaturing con-

325


326

280

290

300

WAVELENGTH (nm)

Fig. 5. Difference spectra of the inhibitor produced in the presence of urea. Protein concentrations used were 1.46 mg/ml. I, in 2 M ; II, in 4 M ; III, in 8 M ; IV in 8 M urea at 100° for lOmin; V, 10-fold dilution of IV.

trum in 3 M guanidine hydrochloride showed positive peaks at 285 and 294 nm, while in 6 M guanidine hydrochloride and in 6 M guanidine hydrochloride at 100° for 10 min the inhibitor showed extremely deep troughs at 290 and 280 nm, ascribed to the exposure of buried tryptophan and tyrosine residues to the solvent. However, the residues exposed to the solvent were immediately masked upon dilution of the denaturant, as shown in Fig. 6. These data suggest that the inhibitor has a remarkable ability to renature reversibly. These data are in agreement with the results from CD spectra. Spectrophotometric Titrations of the Inhibitor—Figure 7 shows the results of spectrophotometric titrations. The phenolic hydroxyl groups were ionized above pH 10.5. The forward titrations showed that 4.4 residues immediately and 5.0 residues after 3 hr, accounting for all the residues in the inhibitor, were ionized at pH 12.9. Although 4 tyrosine residues are rapidly titrated, the last one is ionized above pH 12.5. Since the inhibitor is denatured above pH 12.5, the last tyrosine residue appears to be titrated as a result of exposure of the residue above this pH. The ionization of the last residue is probably irreversible and time-dependent. Cleavage of Disulfide Bridges attd Carboxymethylation of Sulfhydryl Groups—The reduc-

0.4 0

]/

0.3

b

300

WAVELENGTH (nm)

Fig. 6. Difference spectra of the inhibitor produced in the presence of guanidine hydrochloride. Protein concentrations are the same as in Fig. 5. I, in 3 M ; II, in 6 M; HI, in 6 M guanidine HC1 at 100° for 10 min; IV, 10-fold dilution of III.

o

O

5

0.1

290

UJ

J 1

0.2

280

3 O

i

..A

8

o

9 10 11 12 13 PH

Fig. 7. Spectrophotometric titration curves of the inhibitor. Native inhibitor in 50 mM phosphate buffer (pH 7.0) was titrated in a cuvette with 1.5 N NaOH. Titration was carried out within 10 min ( • ) and after 3hr (O). / . Biochem.


urea at 100° caused the disappearance of these troughs, suggesting that tryptophan and tyrosine residues exposed to the solvent by denaturation in 8 M urea at 100° were immediately masked upon removal of the denaturant. The difference spectra of the inhibitor in the presence of guanidine hydrochloride vs. native inhibitor are shown in Fig. 6. The spec-


i /

-20

200

220

240

WAVELENGTH (nm)

Fig. 8. Far-ultraviolet CD spectra of native and reduced and carboxymethylated inhibitor. I, native inhibitor; II, reduced and carboxymethylated inhibitor ; III, RCM-inhibitor in 6 M guanidine HC1.

tion of disulfide bridges and the carboxymethylation of sulfhydryl groups produced were carried out with mercaptoethanol and iodoacetic acid (10), and the effects of the modification on the inhibitory activity and the conformation were studied. The modified inhibitor was found to have neither inhibitory activity nor any cystine residues in the molecule, as determined by an amino acid analyzer. The CD spectrum of RCM-inhibitor is shown in Fig. 8. The depth of the trough at 222 nm was significantly reduced, the helical content was decreased to 14.2%, and the trough at 200 nm due to random coil structure appeared, although a small amount of secondary and tertiary structures, which may not be involved in the inhibitory activity, was found in the modified inhibitor. In the CD spectrum of RCM-inhibitor in 6 M guanidine hydrochloride, no peaks were observed over the range from 217 to 250 nm. This indicates the destruction of the helical and jS-structure by cleavage of the disulfide bridges in the molecule and by the action of guanidine hydrochloride. The results obtained lead us to conclude that the disulfide bridges of the inhibitor contribute to the structural stability and reversibility of renaturation of the inhibitor. DISCUSSION As a result of a study of the effects of denaturants, such as urea and guanidine hydroVol. 79, No. 2, 1976

chloride, and heat on the inhibitory activity, it was found that no decrease in the activity was caused by these denaturants. In order toclarify these unusual properties, the conformational properties of the inhibitor have been studied using both physical and chemical techniques. The inhibitor was found to have an ordered structure, approximately 41% a-helix, 25% /3structure, and 34% unordered structure, by means of CD measurements (Fig. 1). It was shown that the structure of the inhibitor was surprisingly resistant to 2—8 M urea, 3 M guanidine hydrochloride and heat, and was also considerably resistant to hot 8 M urea (Figs. 2 and 3), although under the latter conditions its conformation was partially unfolded. The conformation of the inhibitor, however, was considerably unfolded by both 6 M guanidine hydrochloride and 6 M guanidine hydrochloride at 100° (Fig. 3), and the helical contents decreased to about 13.5 and 10.2%, respectively. When these denaturants were diluted 10- or 30fold with water, the conformation was significantly restored, to 29% ar-helix, indicating that the conformational change of the inhibitor has unusual reversibility. The 29% helical structure reformed may be important for the inhibitory action, since this helical content was nearly the same as the content obtained by 10- or 30-fold dilution after 8 M urea and heat treatment. The difference spectra of the inhibitor in 8 M urea at 100° or in 6 M guanidine hydrochloride referred to the native inhibitor had peaks at 294 and 285 nm and troughs at 290 and 280 nm. The peaks and troughs, which were probably due to the exposure of tryptophan and tyrosine residues buried in the molecule to the solvent, immediately disappeared after dilution of the denaturants. The restoration of the conformation agreed well with that determined from the CD measurements. To clarify the mechanism of the unusual stability of the conformation, the three disulfide bridges in the molecule were investigated. The disulfide bonds were completely reduced and carboxymethylated. The RCM-inhibitor obtained, which had no inhibitory activity, was found to have greatly reduced ordered struc-


n /

-10

327

328


/ . Biochem.


ture and to have only 14% a-helix. This sug- The authors would like to thank Dr. Kashiwatnata gests that the conformation related to these and the Aichi Prefectural Colony Center for CD and disulfide bridges is probably essential for the ORD measurements. inhibitory activity. Vincent and Lazdunski (12) have shown that the integrity of the REFERENCES Cysn-Cysas bridge in pancreatic trypsin in1. Vincent, J., Chicheportiche, R., & Lazdunski, hibitor is essential for a very tight association M. (1971) Eur. J. Biochem. 23, 401-411 with trypsin. It has also been found that 2. Mikola, J. & Suolinna, E.M. (1969) Eur. J. cleavage of all the disulfide bonds completely Biochem. 9, 555-560 inactivates pancreatic trypsin inhibitor (13, 3. Ogiso, T., Noda, T., Sato, Y., Kato, Y., & 14). The existence of a disulfide loop in lima Aoyama, M. (1975) / . Biochem. 78, 9-17 bean trypsin inhibitor was also suggested by 4. Folin, O. & Ciocalteau, V. (1927) / . Biol. Chem. Krahn and Stevens (15). 73, 627-650 The inhibitor activity was also found to 5. Sugiura, M., Ogiso, T., Takeuchi, K., Tamura, S., & Ito, A. (1973) Biochim. Biophys. Ada 328, have unusual resistance to alkaline pH, al407-417 though above pH 12.5 the inhibitor was par6. Cassim, J.Y. & Yang, J.T. (1969) Biochemistry tially inactivated. This result is supported by 8, 1947-1951 the CD spectra at alkaline pH; the spectra 7. Hamaguchi, K. & Takesada, K. (1971) in below pH 12 are almost the same as that of Tanpakusitsu no Senkosei pp. 28-70, Univ. of the native inhibitor and in 0.1 M NaOH are Tokyo Press, Tokyo only partly altered (Fig. 4). As a result of 8. Holzwarth, G. & Doty, P. (1965) / . Am. Chem. the spectrophotometric titrations, it was found Soc. 87, 218-228 that the non-ionized phenolic hydroxyl groups 9. Sherman, M.P. & Kassell, B. (1968) Biochemistry in the inhibitor were ionized above pH 10.5, 7, 3634-3641 and the ionization of one tyrosine residue in- 10. Crestfield, A.M., Moore, S., & Stein, W.H. duced above pH 12.5 appears to be due to ex(1963) / . Biol. Chem. 238, 622-627 posure of the residue to the solvent as a re- 11. Greenfield, N. & Fasman, G.D. (1969) Biochemistry 8, 4108-4116 sult of denaturation of the inhibitor. The fact that the inhibitor has a significant resistance 12. Vincent, J. & Lazdunski, M. (1972) Biochemistry 11, 2967-2977 to alkaline pH is probably attributable to the 13. Dlouha, V., Pospisilova, D., Meloun, B., & Sorm, presence of the disulfide bridges, since even F. (1965) Collection Ctech. Chem. Commun. 30, at pH 13 (0.1 N NaOH) a large part of the 1311-1325 native conformation was retained. 14. Kassell, B., Radicevic, M., Ansfield, M.J., & Based on these results, it seems likely that Laskowski, M., Sr. (1965) Biochem. Biophys. Res. the inhibitor molecule contains a rigid disulfide Commun. 18, 255-258 loop, and the disulfide bridges contribute to the 15. Krahn, J. & Stevens, F.C. (1970) Biochemistry 9, 2646-2652 structural stability of the protein and are involved in the reversibility of conformational change. Barley trypsin inhibitor thus provides an example of the reversible destruction of •secondary and tertiary structure.

Effects of dimethyl sulfoxide on Tetrahymena pyriformis GL. Fine structural changes and their reversibility.

Structural changes in heart and vessels during hypertension with aspects on their reversibility.

Studies on soybean trypsin inhibitors. XI. Complete amino acid sequence of a soybean trypsin-chymotrypsin-elastase inhibitor, C-II.

Tumor-associated trypsin inhibitor in induced and acquired immunodeficiency. Studies on transplanted and HIV-infected patients.

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13C NMR studies of the binding of soybean trypsin inhibitor to trypsin.

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Hypertrophy and hyperplasia of the neonatal rat exocrine pancreas induced by orally administered soybean trypsin inhibitor.

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Structural and metabolic changes in articular cartilage induced by iodoacetate.

Human pancreatic secretory trypsin inhibitor.

Reversibility of joint changes produced by immobilization in rabbits.

Studies on nitrite reductase in barley.

Protective effect of human pancreatic secretory trypsin inhibitor on cerulein-induced acute pancreatitis in rats.

Structural changes in actomyosin induced by Ca2+ ions.

Urea-induced structural changes in chromatin obtained by sedimentation.

Structural changes of linear DNA molecules induced by cisplatin.

Molecular cloning and sequence analysis of the cDNA encoding the human acrosin-trypsin inhibitor (HUSI-II).