Ultracentrifugal analysis of seminal ribonuclease

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To summarize our findings, these results appear completely analogous to those reported on collagen hydroxyproline formation1, 2. Therefore, it m a y be assumed that the mechanisms involved in the hydroxylation of proline to hydroxyproline and of lysine to hydroxylysine are essentially the same, especially with regard to metal ion participation. The authors wish to thank Prof. W. GRASSMANN and Dr. M. CHVAPIL for valuable advice. The technical assistance of Miss H. RENZ is gratefully acknowledged. J. HURYCH was supported b y a fellowship of the Max-Planck-Gesellschaft zur F6rderung der Wissenschaften e.V.

Max-Planck-Institut fi~r Eiweiss und Lederforschung, Miinchen (Germany)

JOSEF HURYCH* ARNOLD N O R D W I G

I J. HURYCH AND M. CHVAHL, Biochim. Biophys. Acta, 97 (1965) 361. 2 M. CHVAPIL, J. HURYCH, E. EHRLICHOV~ AND B. ~MUCHALOV,6., Biochim. Biophys. Acta, in t h e press. 3 F. M. SINEX, D. D. VAN SLYKE AND D. R. CHRISTMAN, J. Biol. Chem., 234 (1959) 918. 4 S. M. FITCH, M. L. R. HARKNESS AND R. D. HARKNESS, Nature, 176 (1955) 163. 5 W. TROLL AND J. LI~IDSLEY, J. Biol. Chem., 215 (1955) 655. 6 H. STEGEMANN, Z. Physiol. Chem., 311 (1958) 41. 7 L. STRAUCH, Z. Klin. Chem., 3 (1965) 165. 8 K. HANNIG, Clin. Chim. Acta, 4 (1959) 51. 9 S. MOORE AND W. H. STEIN, J. Biol. Chem., 211 (1954) 907. io J. HURYCH AND M. CHVAPIL, Bioehem. J., 97 (1965) 236.

Received July 8th, 1966 Revised manuscript received November 23rd, 1966 * P e r m a n e n t a d d r e s s : D e p a r t m e n t of E x p e r i m e n t a l Biology, I n s t i t u t e of I n d u s t r i a l H y g i e n e a n d O c c u p a t i o n a l Diseases, Prague, Czechoslovakia.

Note added in proof (Received April 27th, 1967) PROCKOP, WEINSTEIN AND MULVEN¥ (Biochem. Biophys. Res. Commun., 22 (1966) 124) c a m e to t h e s a m e conclusions r e p o r t e d here, u s i n g a different s y s t e m .

Biochim. Biophys. :4cta, 14o (1967) 168-17o

BBA 33022

Ultracentrifugal analysis of seminal ribonuclease Seminal ribonuclease is an enzyme, isolated from bull seminal plasma, whose general properties are similar to those of the pancreatic ribonuclease isolated b y Kunitzl, 2. This note presents results obtained in a comparative study of the behaviour in the ultracentrifuge of the seminal and pancreatic ribonucleases. Seminal ribonuclease was prepared according to D'ALESSlO et al. (refs. I, 2 and manuscript in preparation). Five different preparations were analyzed, whose specific activity varied between 18 and 33 Kunitz units. In fact the highest specific activity values were obtained in the last two preparations in which a new purification step was added. Pancreatic ribonuclease was the commercially available Sigma 5 × crystalBiochim. Biophys. Acta, 14o (1967) 17o-173

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171

lized, salt-free, type I × A enzyme, containing 9 o % A and I0 % B. Concentrations were calculated from the absorbances measured at 278 m~ b y taking the extinction coefficient xCE - - II% cm/~ as 5.20 for seminal ribonuclease and 6.70 for the pancreatic enzyme, and from the areas of the schlieren peaks; the two sets of results were in good agreement. The experiments were performed in a Spinco model E ultracentrifuge equipped with a rotor temperature indicator and control unit. The sedimentation velocity runs were performed at 42040 and 59780 rev./min in o.05 M sodium phosphate buffer at p H 7.0. Sedimentation coefficients were evaluated from the movement of the m a x i m u m ordinate of the peak using a least-squares procedure, and were reduced in a conventional way to s2o,w. The diffusion coefficients were calculated from the areas of the schlieren diagrams in the sedimentation experiments and corrected for radial dilution and for the movement of the boundary in the centrifugal field according t o ELIAS a.

Molecular weights (M) were obtained from Sobs D e o r r relationship M-TABLE $20,w,

on

the basis of the

RTs D (I --Vp)

I

D20,w

AND

M

OF

SEMINAL

AND

PANCREATIC

RIBONUCL~ASE

IN

0.0 5 M

SODIUM

pHOSPHATE

BUFFER AT p H 7 . 0

Sample

Conch. (mg/ml)

Temperature S2o,w × 1013

D2o.w

Seminal ribonuclease Prep. I 2.70 4.7 ° 6.90 8.90 I 1.8o 18.4° Prep. 2 5.6o 8.6o 11.7o Prep. 3 2.5o Prep. 4 4 .80 4.80 5.4 ° 5.4 ° Prep. 5 4 .80 4.80 2.40

I5.OO 15.oo 15.oo 15.oo 15.oo 15.°° 14.9o 14.9o 14.9o 5 .o0 15-55 13.55 14.25 14.75 14.75 14.5 ° 7.30

8.8 8.0 9.0 8.1 8.2 7.7

25 26 24 26 25 25

800 7o0 6oo 5oo 400 ooo

s/D s/D s]D s/D s/D s/D

7-9

28 2o0

s/D

23 850

Archibald*

24 26 25 23

Archibald*

Pancreatic ribonuclease 4.6o 6.1o 8.50 14.9o 7.90 7.90

14.9o 14.85 14.9o 14.9o 15.8o 15.8o

2.71 2.57 2.64 2.58 2.48 2.30 2.60 2.55 2.55 2.66 2.64

M

Method

× I0 ?

2.60 2.64

1.8o 1.78 1.78 1.77

8.3

3o0 7o0 50o 2o0

s/D Archibald** Archibald**

lO.49

13 800

s/D

14 70o 14 ooo

Archibald**

lO.5O

s/D

* R o t o r speed 17250 rev./min. ** R o t o r speed 23 15o rev./min.

]9iochim. Biophys. Acta, 14o (1967) i 7 o - i 7 3

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The value of ~ = o.711 was calculated on the basis of the amino acid composition of seminal ribonuclease and is similar to that reported in the literature for pancreatic ribonuclease 4. Direct measurements of the molecular weight were also obtained in a few experiments by the Archibald method 4 using the meniscus only; these runs were made both at 1725o and 2315 ° rev./min. The sedimentation coefficients, diffusion coefficients and molecular weights obtained for seminal and pancreatic ribonuclease are reported in Fig. i and Table I. Both proteins sediment as a single, symmetrical peak (Fig. 2); Prep. 2 of the seminal I

o

~0 1

t

0

t

0.5

I

1.0

1-5

2-0

Protein concn. (mg/ml) Fig. I. s20.w as a f u n c t i o n of p r o t e i n c o n c e n t r a t i o n . O, s e m i n a l r i b o n u c l e a s e ; 0 , p a n c r e a t i c ribonuclease. Solid lines were c a l c u l a t e d w i t h a l e a s t - s q u a r e s procedure. Fig. 2. S e d i m e n t a t i o n v e l o c i t y p a t t e r n of s e m i n a l (bottom) a n d p a n c r e a t i c (top) ribonucleases. P h o t o g r a p h t a k e n 94 min after r e a c h i n g the full speed of 59 78o r e v . / m i n . TABLE II S20 O F

SEMINAL

AND PANCREATIC

RIBONUCLEASE

IN

DIFFERENT

EXPERIMENTAL

CONDITIONS

The s e d i m e n t a t i o n v a l u e s are corrected o n l y for t e m p e r a t u r e .

Ribonuclease

Concn. (mg/ml)

Buffer

Temperature S2o × lO 13

Pancreatic Seminal Pancreatic * Seminal *

6.o0 5.5 ° 6.0o 5.5 °

NaHCO 3 + NaOH, I = o.i, p H = i o

lO.25 lO.25 13.6o 13.60

1.72

Pancreatic

6.2o

C H s C O O H + CH3COONa, I = o.I, pFI = 3.8

5.80 6.20 5.8o

9.4 ° 9.4 ° 7.55 7.55

1.4o

Seminal Pancreatic* Seminal" Pancreatic

6.4o 5.8o 6.4o 5.8o

15.75 15.75 lO.3O lO.3O

1.48

Seminal Pancreatic* Seminal" Pancreatic Seminal

4.oo 2.5 °

*

6 M u r e a + o.o5M Tris + o.I M KC1

2 M guanidine-HC1

14-2o h a f t e r t h e first run.

Biochim. Biophys. Acta, 14o (1967) 17o-173

lO.5O lO.5O

s20 × 2:013

2.53 1.70

2.42

2.10

1.48 2.13

2.16 2.37 2.66 o.61 1.21

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enzyme, however, showed about IO % of a slower component. The results obtained for pancreatic ribonuclease (s°20,w = 1.75 and M ~ 14200) are in good agreement with those reported in the literature 5. Seminal ribonuclease studied under identical conditions behaves differently; the value of s°20,w is 2.73 and the molecular weight (25 5o0) is about twice that of the pancreatic enzyme. This might be explained b y assuming that the seminal enzyme consists of only one polypeptide chain, twice the length of that of the pancreatic enzyme or, alternatively, of two chains similar to the pancreatic enzyme chain. A few attempts were made to determine whether the seminal enzyme dissociates under conditions which are known to promote the splitting of proteins into subunits. The results obtained from parallel experiments on seminal and pancreatic ribonucleases (the latter as a control) are shown in Table II. Clearly no dissociation of the seminal enzyme is evident under any of the conditions used. Thus, if the seminal ribonuclease is made up of two subunits, it must have a great stability with respect to dissociation. That the chains are linked by covalent bonds seems somewhat unlikely~. Chemical and physicochemical investigations to test these alternatives are in progress. We are grateful to Dr. E. A~TONINI for helpful discussions during this work. This investigation was partly supported b y research grant N.M 64.116 from the New York Population Council to A.F., G.D.A., and E.L.

Institute of Biological Chemistry, University of Rome, C.N.R. Centerfor Molecular Biology, Rome (Italy) Institute of Biological Chemistry, University of Perugia, C.N.R. Impresa di Enzimologia, Perugia (Italy) I 2 3 4 5 6

G. G. H. H. H. H.

LUCIANO FORLANI EMILIA CHIANCONE PAOLA VECCHINI ARDESIO FLORIDI GIUSEPPE D'ALESSIO

ENzo LEONE

D'ALESSlO AND E. LEONE, Biochem. J., 89 (1963) 7 P. D'ALESSlO AND E. LEONE, Proc. Ist Meeting Federation Enzymol. Biochem. Soc., A25 (1964). G. ELIAS, Ultrazentrifugen-Methoden, Beckman Inst., GmbH Miinchen, 1961. K. SCHACHMAN,Methods in •nzymology, Vol. 4, Academic Press, New York, 1957, p. 33A. SCHERAGAAND J. A. ROPLEY, Advances in Enzymology, 24 (1962) 161. K. SCHACHMAN,Cold Spring Harbor Syrup. Quant. Biol., 29 (1964) 4o9.

Received February 2oth, 1967 Biochim. Biophys. Acta, 14o (1967) 17o-173