The haemoglobins of Artemia salina II. Isolation of three haemoglobins

Int. 3* Bidem., 1970,I[,537-545

537

THE HAEMOGLOBINS II.

WARING,

GAIL Department

ISOLATION

OF ARZ’Y34.Z” SAL.I.,‘V’A

OF THREE

MAN-CHIU

POON,

HAEMOGLOBINS

AND SARANE

T. BOWEN

of Cell and Molecular Biology, San Francisco State College, San Francisco, California 34132, U.S.A. (Rccektrd 25 Nov., 1968) ABSTRACT

I. In the haemolymph of the brine shrimp, Arkar&~sufkz, are 3 haemoglobins: Hb-r, Hb-2, and Hb-3. Each has been isolated from a whole animal homogenate by ammonium sulphate precipitation, DEAESephadex chromatography, and G-200 Sephadex gel f&ration. 2. In poiyacrylamide disk clectrophoresis, Hb-I and Hb-2 are single bands whereas Hb-3 has 3 bands, The relative densities of the 3 bands shift with changes in concentration, suggesting an association-dissociation phenomenon for Hb-3. 3. The 3 haemoglobins have the same sedimentation coefficient (I I -3S,, ,), which suggests that they have similar molecular weights.

THE

haemolymph

of

the

brine

shrimp,

Artmiiasdiiaa, cont.ains 3 haemoglobins : Hb-1 , Hb-2, and Hb-3 (defined by their mobilities in cellulose polyacetate electrophoresis). This paper outlines a method of isolating each of the 3 haemogiobins from a

whole animal homogenate and describes their polyacrylamide disk electrophoresis patterns and rates of sedimentation on sucrose density gradients. MATERIALS

AND METHODS

During the summers of x967-8, 24 batches of Artem& were collected from salterns of the Leslie Salt Company on San Francisco Bay. In some lots, all shrimps were red; in others, they were mixtures of pink, green. and blue. All had matured beyond the seventh instar but some were 5-g mm. and others Q-I I mm. in size. Haemoglobinwas present in all batches, Hb-3 was detected in IO, whereas Hb-x was present in only 2 batches. Artrmia were brought directly from the salterns into the cold room where feathers and debris were removed. They were netted through 3 successive washes in clean tap water and drained in a net. PURIFICATION The Tris-Cl buffer used throughout this study was o-50 M Tris. ionic strength o-04. pH 7.5, at 25’ C. Ion-exchange chromatography was carried

DEAE-Sephadex A-50 coarse (Pharmacia, Uppsala, Sweden) washed through 3 cycles with an excess of buffer (o- 125 M NaCl in TrisCl). Agarose A-t5m gels were from Biorad Laboratories, Richmond, California. G-15, G75, G-100, and G-loo Sephadex gels (Pharmacia) were hydrated in boiling water, defined through 6 cycles, and equilibrated with Tris-Cl buffer. Columns were developed by gravity flow. Absorbance of selected fractions was determined at 260, 280, 415, and 540 rnp in a Zeiss M4QIII spectrophotometer. NaCl gradients were inferred from ~onductivities mea&red by a Beckman RC-r6B2 bridge. Protein soiutions were concentrated by dialysis against Tris-CI buffer under pressure (Huehns and Shooter, I 961). All purification procedures were carried out at 5’ C. The ammonium sulphate precipitate was sometimes stored under loo per cent saturated ammonium sulphate. All other fractions were concentrated by pressure dialysis against Tris-Cl but&r and stored in the freexer (-20” C.). out on.

EsTExtAsEASSAVS Esterase activity as a measure of proteolytic enzyme content was assayed using b-toluenesulphonyl-L-arginine methyl ester (TAME) as a substrate. When TAME Determatubes (Worthineton Biochemical Corp., Freehold, NJ.) we% reconstituted with 6.0 ml. of water, the substrate solution was 8.7 x IO-* TAME in oo5 M phosphate, pH 8.0. TAlME hydrolysis (producing methyl alcohol and tosyf arginine) was followed by spectrophotometric monitoring of increase in absorbance at 247 rnfl due to increase in tosyl

WARING

538

arginint. Esteram activity is defined as increase in absorbance at 247 mp per minute divided by absorbance at 415 mu. Runs were carried out as described by Smithies

( 1g5g),

with

the

modifications

of

Gammack,

Huehns, and Shooter (1960) using Tris-EDTAborate buffers of pH 8.3 (Roscmcyer and Huehns, 1967) in 12 per cent atarcb. The buffer stock solution (69.9 g. Tris, 5.84 g. disodium EDTA, and 3og g. horic acid per 1.) was diluted I to 20 for making the gel and I to 5 for the Runs were made at buffer chambers. 20 mA. constant current for 3 hours at 4” C. Gels were sliced into two parts which were stained with Buffalo Black NBR or ortho-dianisidine. CELLULOBEPOLYACZETATE E~JKTROPHORESIS Rum were carried out by the method of Kohn (1g6o) at 27 V. per cm. for 3645 minutes at 25’ C. on Senranhore III wlvacetatc (Gelman

I&trummts ‘Co:) in barbital bufIcr ’ (0.92 g. diethylbarbituric acid and 5. I 3 g. sodium barbital per litre; pH 8.6). Strips WCFC atined in 0’2 per cent Ponccau Red S in fixative (7.5 per cent sulphosalicylic acid&g per cent trichloro-acetic acid) and ~dcatained in - 5 per cent acetic acid. Relative mobilitia of the 2 Arti hacmoalobins have been illustrated car&r (Bowen, Lgbhera, Poon, Chow, and Grigliatti, 1g6g). POLYACR-

E

DrascEXYKYROPHO~~~~~

Disk elcctrophoruis

followed

the method

of

Davis (1964) but used o-47 x 123 cm. tubes and Tris-glycine buffer with resolving #H of 9.6 at 25” C. and 1~3 at o0 C. (Varon, Nomura, and Shooter, 1g68). The resolving gel was made by mixing A, C, and P solutions in theratio x:1:2. The solutions had the following composition : Sdution A: 24 ml. of I X HGI, r8srg g. Tris, 0.4 ml. Tuned (N,&V’,W-tetmmethylethylcncdiamine, Eastman 8178) per tooml.; Solution C: 3og. acrylamide (Eastman 5521) and ~8 g. Bis (N,N rne~yl~eb~~~d~ Eastman 8383) per too ml. ; Solution P: 300 mg. ammonium persulphate per too ml. By this method, 7’5 per cent gels were prepared. To make 4 or to per cent gels, the amounts of Bii and acrylamide in Solution C were varied proport.iotlatcly. The stacking gel was made by mixing B, D, and E solutions in the ratio I

: 2 : I.

The

soluti~us had the following com-

position: SolutionB: t2*8ml.of I MH,PO,, 2.85g. Tris,andoxoml.Tcmcdpcrrooml.;SolutionD: 5 g. acrylamide and 1.25 g. Bii per tooml.; Solution E: 2 mg. riboflavin (Nutritional Biochemical Corp.) per xooml. Upper (cathode) buffer was 6-42 g. Tris and 398 g. glycine per litrc. Lower b&m was I 2.1 g. T&t and 50 ml. of I N HCi per litre. Samples contaiuing o.o2+4o unit absorbance 415 mu were diluted to a vohtme of 5c+roo 4. with Tris-Cl buf%r (PH 75, ~04

ET AL.

Int. J.

3iochm.

ionic strength) and sucrose was added to a final concentration of 5 g. per xoo ml. (The AIIJ unit is defined as absorbance at 4x5 rnt,t xml. of

sample.) The current was o-25 mA. per tube as the samples entered the stacking gel. After they entered the resolving gel, the current was increased to I mA. per tube. Gels were stained in 0.1 per cent Amid0 black (naphthol blue black NBR. Allied Chemical1 in to ncr cent acetic acid for I hour, 2nd destain&l overnight in IO per cent acetic acid. An alternative procedure was fixation for 30 minutes in 20 per cent trichloro-acetic acid, staining in 0.1 per curt Coomassie blue (Coomassie briBiant blue R-250, Golab Laboratories, Inc.) in 20 per ccut trichloro-acetic acid for I hour, destaining overnight in ro per cent trichloro-acetic acid. ELECTROPWORETKBL~JTXON OF PROIXIN BANDS FROMUN~XUNXD GEU Laxgc pore gels (4 per cent acrylamide, 0.4 ml. volume) were photopolymerized in 0’47 cm. (i.d.) glass tubes, then removed and placed in 0.55 cm. (i.d.) glass tubes constricted at one end. A bag

of cellulose dialysis tubing (o3g cm. flat width) 8llcd with Tris-Cl buf%r was attached ta the constricted end. Two or three gel fragments containing unstained protein bands were placed on the large pore gels and elutcd into the dialysis bag by &ctrophorcsis at constant voltage (300 V.I. Eluted proteins were concentrated by prcssttre dialysis against T&Cl buffer. ULTRACENTRW~~ATIONIN SUCROSEDEN~~TY

GRAntENZ#

For separation of haunoglobins from PL protein, the 0.2~ml. sample (I per cent protein) was layered on a 4-ml. iin& &rose gAiient’ 20 uer cent w/v sucrosc~ in Tris-Cl buffer.

(xo-

For d&rmination ‘of sedim&tation coefficients, the O’I-ml. sample (o-2+5 per cent hacmoglobin) was layered on a 4-ml. gradient (5-20 per cent sucrose) in Tris-Cl buf%r (pH 7.5) or glycine-NaCl buffer (pH IO, ionic strength o’ IO) prepared from data of Bates ( I 968). Gradiaus were cmtrifiigcd in a SB-qog rotor in the International Equipment Co. Model B-60 for 8 hotus at 55,000 r.p.m. at 5” c. The contcn~ of each tube were colkxted in 3o-40 fwtions and absorbance determined at

280 mu 2nd 415 mu on a photometer. Approximate culated by the method ( I g6x ) with no corrections RESULTS

Zeiss M.,+QIII spcctroS,, . values were calof Martin and Ames for concentration.

AND DISCUSSION

PROCXDURE FOR IsfXAllON

OF

3 HAEbiO-

GLOBINS Outline ofPr5~e~~ The purification steps arc shown in T&e 1 (flow sheet) and Fig. I (~l~c~~de disk

HAEMOGLOBINSOF ARTEMIA

1970, 1

electrophoresis assay of fractions). The most common residual impurities in haemoglobin preparations were the colourless PL protein and the green proteins described earlier (Bowen and others, I 969). Because the concentrations of the 3 types of haemolymph proteins (haemoglobins, PL, and green proteins) fluctuate in response to the environment, each batch of Astemia had a different composition. Therefore, the number of columns needed to process one 4oo-ml. batch was not always the same. If the ammonium sulphate

539

The ratio of .4s,,,, to Alis ranged from I ‘4 to 2.6 for the filtered supematant of the homogenate, dropped to 06-r *I for the 50 per cent ammonium sulphate precipitate, 0.37-o-39 in the DEAE-Sephadex stepwise fraction, o.32-o.3g for the DEAE-Sephadex linear gradient fraction, and 0.28-0.30 for the G-200 eluate. The & : A,, ratio ranged from I so to 0.8 throughout the procedure. Extraction of Proteins

Two volumes of drained shrimp were added to I volume of Tris-Cl buffer and

Table Z.-FLOW SHEET FOR ISOLATION OF 3 drtemia HAEaaooLosms Procedure

step Jvo. I

2

3

4oo ml. drained drtcmiu and 200 ml. Tris-Cl buffer (~JH 7.5, ionic strength 0.04) were homogenized in a blendor, then centrifuged at 27,000 g for 30 minutes to give SUPERNATANT. This was filtered through No. I Whatman paper to remove the yellow lipid layer and give 3oo-400 ml. FILTEREDSUPERNATANT. To the filtered supernatant, solid ammonium sulphate was added to 50 per cent saturation. Centrifugation at 27,000 g for 30 minutes gave 15-25 ml. PRizuprrA~. A 4-ml. portion of precipitate was dissolved in Tris-Cl buffer to give total volume of I I ml. loaded on a G-25 Sephadex column (I no-ml. bed volume) equilibrated with o* 125 M P3aCI in T&-Cl buffer and eluted in the same buffer to give 2o-25 ml. DESUTED PRECPITATE.

4

The desalted precipitate was loaded on to a DEAE-Sephadex A-50 column (12 x 2.5 cm.) equilibrated with o I 25 M NaCl in T&Cl and washed with xoo ml. of the same buffer. The haemoglobin was eluted with 150 ml. of 0.225 M NaCl in Tris-Cl (initial flow-rate of 12 ml. per hour per sq. cm.) to give DEAE ~TEPWLWEL~JTIONFRACTION. This was equilibrated by pressure dialysis with T&Cl buffer.

5

Three DEAE-Sephadex stepwise elution fractions (each derived from 4 ml. precipitate from Step 2) were pooled and loaded on a DEAE-Sephadex A-50 column (12 x 2.5 cm.) equilibrated with 0.125 M NaCl in Tris-Cl, washed with loo ml. of the same buffer and eluced by 400 ml. of a linear NaCl gradient (0. t25-0’225 &f, with a flow-rate of less than 8 ml. per hour per sq. cm.) to give DEAE gradient elution fractions. The haemoglobin fractions were concentrated by pressure dialysis against T&Cl buffer, checked by cellulose polyacetate electrophoresis, and those containing only I haemoglobin were the CONCENTRATED DEAE GRADIENT ELUTIONFRCTIONS which contained either Hb-3 (first off column), Hb-2. or Hb-x (last off column).

6

Each concentrated DEAE gradient fraction was applied to a G-noo column (36 x2-5 cm.) equilibrated with T&Cl buffer and eluted with the same buffer at a flow-rate of 2 ml. per hour per sq. cm. The eluted G2oo FRACTION was concentrated by pressure dialysis against Tris-Cl buffer and stored in a freexer (-20' C.). From 4oo ml. of Artemio, xoo-450 mg. of haemoglobin were recovered.

precipitate was 25 ml. (Step 2 in Table I), 6 G-25 columns, 6 DEAE-Sephadex stepwise elution columns, 2 DEAE-Sephadex linear gradient columns, and 2 G-200 Sephadex columns were needed to process the haemogiobins from 400 ml. of drained shrimp.

homogenized (in 50-n& aliquots) in a semimicro container of a Waring Blendor for less than I second. Particulate matter was sedimented in a Sex-vail RC-2 refrigerated centrifuge at 27,000 g (max.). On top of the supernatanr (which varied from red to brown) was

540

WARINGET AL.

a yellow lipid layer. This adhered to the paper when the supematant was filtered through Whatman No. I paper. Ammonium Sulphatc Precijdation Solid ammonium sulphate (3 12 g. per 1.) was added to the filtered supematant to bring it to 50 per cent saturation. No attempt was made to control PH. After stirring for 30 minutes followed by centrifugation at 27,000 g for 30 minutes in the Servall RC-2,

FIG. I .-Polyacryhunide disk electrophoresis patterns of fractions from purification of Hb-z.

Tris-glycinate buffer system, 7+ per cent gels, resolving pH lo-3 at o” C., run at 4” C., stained with O’I per cent Amid0 black (details in

Materials and Methods). The amount of haemoglobin on each gel is o21-o28 Alls units. (All, unit =A,,, x ml. of sample). Migration for 6 hours at I mA. per gel. A, Filtered supernatant of whole animal homogenate, containing Hb-I, Hb-2, and contaminants (e.g., PL and green proteins). 6, 50 Per cent ammonium sulpbate precipitate. C, Haemoglobin fraction fmm DEAESephadex column (stepwise elution). D, Haemoglobin- fraction from DEAF-Sephadex column (linear gradient elution). E, Haemoglobin fraction from G-loo Sepbadex coiumn. the grcm supernatant was discarded, and the haemoglobin precipitate was either stored under xoo per cent saturated ammonium sulphate in T&Cl buffer or it was immediately dissolved, desalted by G-25 Sephadex, and put through the DEAE-Sephadex stepwise elution. The redissolved precipitate is extremely vulnerable to enzymatic decomposition. Two batches of Artiia, one containing Hb-I and Hb-2, the other Hb-2 and Hb-3,

ht. J. Biochem.

were subjected to successive 5 per cent increments of ammonium sulphate saturation. The precipitates were redissolved in T&-Cl buffer and absorbance at 280 and 415 mu were recorded. In both batches, the haemoglobin precipitated between 30 and 50 per cent saturation, whereas some of the green proteins were soluble at 50 per cent saturation. Desalting on G-25 Sephuakc To one volume of the ammonium sulphate precipitate, about r-8 volumes of T&Cl buffer were added to adjust the viscosity of the sample to I ‘5 x that of the buffer. The volume of diluted sample loaded on the G-25 Sephadex column was ho-20 per cent of the column bed volume. DEAE-Sephadex Chromatography The initial stepwise DEAR-Sephadex elution (Step 4 in Table I) with high flow-rate was used to quickly separate the haemoglobins from most of the acidic proteases. The loo-ml. wash removed some material which absorbed at 280 mu. The eluted haemoglobins were dialysed under pressure against T&Cl buffer. A second DEAE-Sephadex elution by means of a linear NaCl gradient was used to separate the 3 haemoglobins. Because none of the 24 batches of shrimp contained all 3 haemoglobins, column profiles showed the separation of Hb-2 from Hb-3 or Hb-2 from Hb-I (Fig. 2). Six DEAESephadex linear gradient elutions were assayed by conductiand absorbance at vity, electrophoresis, 280 and 415 mu. Hb-3 was eluted at o-15O-I 7 M NaCl, Hb-2 at o*~g-o-20 kf, and Hb-1 at 0*20-0~22 M NaCl (Waring, 1970). At 0.225 M NaCI, a narrow brown ring and a broad band of green remained on the column. The brown ring was presumed to be haemoglobin denatured by acidic proteases. Esterase activity was I-fold to lo-fold higher in &actions eluted at 0.2 r-o.30 M NaCl than in those eluted in the range 0.15-o*20 M NaCl. If I-&I was not completely off the column at 0.225 M NaCl, an additional wash of 0.225 M NaCl was used (Fig. 2). The gradient was not carried above 0.225 M because green proteins and proteases were eluted at 0-225-0-250 M NaCl.

?lAEMOOLOBIXS OX?ARTEMXA

‘97% 1

Denatured haemoglobin was sometimes eluted at 0*125-o*145 M NaCl. (The greatest amount appeared in the column profile shown inFig. 2.) It migrated as several bands with mobility less than that of Wb-g on cellulose acetate but with greater mobility than all 3 native haemoglobins on polyacx-ylamide 7.5 per cent gels (Tris-glycinate, @-I g-6). Denatured haemoglobin eluted from DEAESephadex colts consisted primarily of 6.W and 3-4s species.

54'

Additional Puri.ution Steps When a batch of shrimp had a high proportion of PL or green proteins, Hb-I contained traces of both contaminants and Hb-2 contained traces of PL after the G-200 Sephadex filtration. Because the PL protein is only slightly larger than the haemoglobins, little separation was obtained on G-200 Sephadex; complete separation could be obtained by sucrose density cen~~tion. The haemoglobins could be separated from

1.2 0.24

1.0

0 9 .r L"0.8 0 z &

0.22E c?

Donaturac+

0.20 ? O.lSi; zi 0.1b;

0.6 O.WL 43

t" 6 * 0.4

0.12

s 0.2

0.0 EWluont

volume

?t-riI,

z.-Chromatographic separation of Hb-r and Hb-2. DEAE-Sephadcx stepwise clution fraction (130 A,,, units in 5 ml.) was Ioadcd on to a DEAE-Sephadex column < 12 x 2.5 cm.) and ehtted by 6oo ml. T&-Cl buffer, @-I 7-5, with Nail gradient: too ml. of o-125 M NaC1, +XI ml. linear gradient (o~125~-225 MN&I), and IOOml. of 0,225 MNaC1. Flow-rate less than 5 ml. per hour per sq. cm, FIG.

Gel Filtration on G-200 Sephadcx Before samples were loaded on G-loo co1umns, the viscosity was adjusted to less than I ‘5 x that of the buffer (Tris-Cl, PH 7.5). Elution profiles at 280 and 415 mp were obtained for 3 samples containing a mixture of Hb-2, Hb-z, and non-haemoproteins ; for I sample containing Hb-3 and impurities ; and for 2 samples containing Hb-2 and other non-haemoproteins (Fig. 3). For all 6 of these G-200 columns, the 415 mv absorbance gave a single peak with slight asymmetry. There was no evidence of separation of the 3 haemoglobins on G-200 Sephadex.

the heavier green proteins on Agarose A- I 5m (2.5 x 30 cm.; IO ml. per hour per sq. cm. flow-rate), RATIONALE

FOR SEQUENCE

OF ~RIFSCATION

STEPS

The whole animal homogenate contained proteolytic enzymes released when the blendor biades ruptured the intestine and other organs of the shrimp. Esterase activity (increase in absorbance 247 mp per minute/ absorbance 4x5 mp) was 0. I35 in the. 50 per cent ammonium sulphate precipitate. Xfter the precipitate was eluted from the DEAE-

542

WARING

Sephadex

by stepwise eiution (Step 4 in eked haemoglobin showed a Q-fold decrease in esterasc activity. After the precipitate was passed through the G200 column, the haemoglobin fraction showed r5-fold reduction in activity. Sephadex-Gzoo was more effective than Sephadex G-75 or G x00 in separating haemoglobins Corn procedures esterases. The 3 fractionation Table

1)

the

ET AL.

Int. J.

Biochern.

sedimentation on sucrose gradients in the same buffers. An approximate sedimentation coefficient of I I +3S was obtained for Hb- I, Hb-2, and Hb-3 by the method of Martin and Ames (I 961) using as reference proteins bovine serum albumin and bovine gamma globulin (4.4.S and 7o.9, respectively). We conclude that the 3 native molecules have similar molecular weights. When the 3

0.0 I

0

volumu tmll Fro. 3.-Sepbadex G-loo gel filtration of DEAE-Scphadex gradient fraction (containing Hb-2 and non-baem proteins). A x*5-ml. sample (containing 120 A,,, units) was loaded on to a C-200 Sephadcx column (2.5 x 36 cm.) and eluted with Tris-Q buffer, pH 7.5, ionic strength o-04, flow-rate of 2’4 ml. per hour per sq. cm. EtWuont

(ammonium sulphate precipitation, DEAESephadex chromatography, and G-200 Sephadex gel filtration) were placed in different sequences and theAsse : Aals ratio determined each step. AB 3 procedures were needed to bring the ratio to minimum values (0.28 for Hb-2 ; o-31 for Hb-3). G2oo Sephadex gel filtration was put last because of the low capacity of G200 and because it separated the native haemoglobins from smaller denatured haemoglobin kgments. DETZRIUINATIONOF SEDIMEXWATION

CoElvmmTs Each of the 3 haemogiobins was dialysed for 24 hours against Tris-Cl or glycme-NaCl buffers (#H 7.5 and 10’0, respectively) before

haemoglobins were denatured by low or high pH (4.0 and x2*0), each gave rise to 6*BT and 3-d species. fJELATI0NSEIIP

OF THE %USMOGLOBXN

IN POLYACRYLAMIDE

BANJX

ELECTROPHORESIS

PATFERNS .Nommlattm Art-m& haemoglobins designated by number (Hh-I , m-2, Hb-3) have been detected in many wild populations and are synthesized by most shrimps of the Great Salt Lake and San Francisco saltem populations when reared under certain laboratory conditions. Spectrophotometric studies have shown that their ratio of Aeat, to A,,, is o28-o-30. Hb-r , Hb-2, and Hb-3 have absorption peaks

HAESIOGLOBINS

‘97% 1

characteristic of haemoglobins (Bowen and others, I 969). Haemoglobins designated by letters (Hb-X, Hb-Y, and Hb-2) are found infrequently and have not been characterized by spectrophotometry. Therefore, we cannot exclude the possibility that they are non-haemoglobins which bind haem. Hb-Y and Hb2 were seen in only I of the IO batches of saltern shrimps which were examined by disk electrophoresis, In a study of the haemolymph of single shrimps, Hb-X was detected

and polyacrylamide systems (T&k II). Because of electro-osmosis on cellulose polyacetate, relative mobility is defined in Table N as the ratio of distances of 2 proteins from human haemoglobii A rather than &om the pencil line ‘ origin ’ on the paper. On cellulose polyacetate (PH 8.6), Hb-1, Hb-2, and Hb-3 each migrate as a single band and their mobilities are greater than that of human haemoglobm A. In 12 per cent starch (fiH 8.3), Hb-2 and Hb-3 have about the same mobility as human Hb-A. On 7.5 per

h%OBXUTIES OF Artcmia IN 2 ELECTROPHORE~IS Sysnms

Table II.--&~~TIvE HA~~~OGLOBINS

+Oriqin

CELL~JLOSE

Adetnia HMtd00~0Rm

ACETATE?

PoLYAcRYtAMmE*

I-E-1

Hb-z Hb-3 Hb-X Hb-Y Hb-Z

543

OF ARTEML4

1’03 ~8 0’4 ~6

O-29

0.26 o-40, ~36, o-24

I

0’11

c

-

WI6

-

Human Hb A

I

* Tris-giycinatc, 7.5 percent gel, pH ~0’3, ato’ C., run at 4” C. (for deta&~ccMaterialsandMethods). The relative mobility of each Art&a hacmoglobin is defined as A/B, where A and B are the distances of Artemiu hacmoglobin and of bovine serum albumin from the top of the resolving gel.

+ Barbital b&&r, pH8.6, Gelmanpaper. Relative mobility is defined as _4’/%‘. A’ is the distance

between Artemia haemoglobin and the slower human haemoglobin; B’ is the distance between bovine serum albumin and human hacmoglobin A. in a small percentage of Great Salt Lake reared under laboratory conditions where Hb-I, Hb-z, and Hb-3 were detect-

males

able in the haemolymph (Bowen and others, 1969). Therefore, Hb-X may be a genetic variant of one of the 3 common haemoglobins. Because Hb-X, Hb-Y, and Hb-Z were present in such small quantities, it was not possible to run them in both electrophoresis systems. We cannot exclude the possibility that Hb-X (detected on cellulose polyacetate) is equivalent to Hb-Y or Hb-Z (detected on polyacrylamide).

Comparisonof 3 ElectrophoresisSystems Relative mobilities of 5 Astemia haemoglobins are compared in cellulose polyacetate

GB5A + HtrZ Hb’f Mb-3 Hb-? Hb-t FIG. 4.-Two reference proteins, bovine serum albumin (BSA) and human haemoglobin A, were mixed with each of 5 Artemia hacmoglobins for disk electrophoresis on 7.5 per cent polyacrylamide geh in Tris-glycinate system, resoivmg pH 10’2, run at 4” C. (0.2~25 _4,,, unit per gel). Gels were stained in o* I per cent Amid0 black. Migration was for 6 hours at I mA. per tube.

polyacrylamide (pH 9.6 or roqg), haemoglobins have a mobility less that of human than haemoglobin -4. On polyacrylamide, Hb-2 is a single band in 4, 5, 6, 7.5, and IO per cent gels. Hb-3 migrates as I band in 4 per cent and II per cent gels, as o bands on 5 per cent gels, and as 3 bands on 7.5 per cent gels. Hb-I is a single band on 7.5 per cent gels. One batch of shrimp yielded Hb-I (defined as I band on cellulose poiyacetate) which gave more than I band on 7.5 per cent polyacrylamide. cent

Artemia

Because little protein was available, we were unable to establish that the bands were independent proteins. Because of its excellent resolution of denatured haemogiobin and traces of impurities, the 7.5 per cent polyacrylamide disk electrophoresis system was used to monitor the putification procedure. In this system, the ratio of human haemoglobin mobility to albumin mobility is ~63 (Fig. 4f. Denatured As&r& haemoglobin migrates between the fast bands of Hb-3 and human haemoglobin. ,--_-.r-

T

Int.

WARING ET AL.

544

.;--

~ i

o.su


-.--__riq,n

-‘1

,

a2u 0.3u absorbance

.03u 0.1~ at 415 mu)

FIG. 5.--EfTect of protein concentration on Iib-3 electrophoresis pattern on 7’5 per cent gcis, Trisglycinate swtem, resolving FH g-6 at 25’ C., migration d&e 8 hours at I mA. per gel. Stained in 0.1 per cent Amid0 black. Unit of absorbance defined as AIis x ml. of sample.

EDidmGct/z&ttk 3 Bandc

of lib-g are

Biochem.

riboflavin and the same 3-band pattern was observed. Evi&nce that &-a, H&T, I~~~ Proteins

and Hb-<

are

Hb-Y and Hb-Z were present in only one batch of Artemia. Because they were red peroxidase-positive proteins, it is possible that they are haemoglobins. They were eluted from the DEAE-Sephadex linear gradient with Hb-2. To exclude the possibility that Hb-2, Hb-Y, and Hb-Z were an association-dissociation system, the 3 bands seen on disk acrylamide electrophoresis (Fig. 4) were eluted and rerun in the same electrophoresis system. Each ran as one band with the same mobility as before. Further evidence that the 3 haemoglobins were not related is the fact that Hb-2 runs as one band at a range of concentrations (0*4-o~040 Ah1s unit loaded on the gel). Because they migrate more slowly in 7.5 per cent polyacrylamide (Fig. 4), Hb-Y and Hb-Z may have a greater molecular weight or higher disymmetry constant (fub) than the 3 common haemoglobins. ACZCWOWLEDGEXENTS

We thank Profmr Eric Shwter for so generously providing the facilities of his laboratory in the Department of Genetic& sta&ord university Medical Genter. We thank Arthur Piltch, Regina Perez, Andrew Smith, and Keith Borden for valuable suggations throughout the study. This research was supported by NIH Grant ?IE

1 r&5*

Compommts of an Association-Dissociation System When different quantities of Hb-3 were

loaded on to 7.5 per cent gels, the electrophoresis patterns were markedly different (Fig. 5). As the amount of protein decreased, the relative staining intensity of the middle band increased. When the &west band was &ted from the gel and run again on the same electrophoresis system, it regenerated all 3 bands in the normal pattern. When the 2 fast bands were eluted and rerun, each gave rise to both fast bands and a small amount of the slowest band. To exclude the possibility that the fast bands are artifacts resulting from persulphate denaturation, Hb-3 was run on gels photopolymerized with

3.

REFERENCES BATES,

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Kg Word Index: Artemia salina, crustacea, haemoglobins, inducible proteins.