Iron binding by tissue proteins

CLINICA

IRON

W.

BINDING

J. MAcLENNAN

University (Revised

371

CHIMICA ACTA

Medical

manuscript

BY

TISSUE

PROTEINS

AND G. PARKER Unit,

Stobhill General

LEWIS Hospital,

Glasgow,

N. I (Great Britain)

received August gth, 1967)

SUMMARY

Using starch gel electrophoresis and immunotitration it was possible to demonstrate the presence of ferritin and transferrin in protein extracts from human tissues. Ferritin was detected in brain, liver and spleen whereas significant amounts of transferrin were found in liver and kidney. No iron-binding protein was demonstrable in muscle. These findings support previous observations suggesting that the liver is an important source of transferrin. The presence of this protein in kidney would indicate that this organ is also a site for storage or production of transferrin.

Several investigators have suggested that the liver is an important source of the iron-binding serum protein-transferrin. Indirect evidence was provided by Braendstrupl and Mandell who recorded high levels in acute hepatitis and low values in patients with cirrhosis. More recently direct evidence has been given by Asofsky and Thorbecke3 that cultured monkey liver cells utilizing 14C-labelled leucine are capable of synthesising transferrin. In addition to transferrin the liver cells contain other proteins capable of binding iron. These include ferritin and haemosiderin. The former is of importance in the storage of iron, whereas the latter is probably a degradation product of ferritin4. Although ferritin and haemosiderin have been isolated from liver cells, data concerning the qualitative and quantitative presence of transferrin in these cells are not available in the literature. This paper reports the results of an investigation into the titres of transferrin to be found in various body tissues using the techniques of starch gel electrophoresis and agar gel immunotitration. METHODS

Materials I. Intracellular protein extracts (total protein concentration 4-8 g/r00 ml) free of cell debris, and of low fat content, prepared from human brain, kidney, liver, muscle and spleen. of 345 pg/roo ml.) 2. Human serum. (With a total iron binding capacity serum, prepared from goat, Baxter Labora3. Specific human antitransferrin tories. Fe). 4. 59FeC1, of high specific activity (I &/,ug C&n. Chim. Acta,

18 (1967)

371-376

Pvoccdwes

I. Pvejwvntiotl of iztvacell&v extuncts. Fresh cadaver tissue (4 8 11post mortem) fineiy divided by mincing, was washed thoroughly six to Q-lit times in normal saline using centrifugation. \\‘ashing \vas continued until the suprnatant \vas niwroscomixture (I :4) wa5 pically free of all but tracts of haemoglobin. A final tissue-saline then homogenized by processing consrcutivcly through three t>pxs of homogeniscr, nan1ely a Kcnmix high speed cutting homogenizer, a Silverman medium spwd shearing homogeniser, and a Griffith’s glass hand grinding tube. (‘cllular debris and free fat were wparatcd from the protein t,xtract 1)~ centrifugation at 5” for 1 Ii at 750 g. The prot~iti-containing sup-natant \ws concentrated l,Jr tlial!5is against cr~stallinc canv sugar at 3” for 24 IiS. This conccntratv was then furtlwr wntt-ifugetl under conditions similar to tile above for a period of 00 min. i\n estimate of thtl total protein concentration in the final supvrnatant \v;i5 made using a niodifivation of thr Eiuret

method6. 2. Stavch gel electvojdzovesis. 2 pu(: of 591Te(‘l, in 0.2 ml of dcionizcd k\-atcr \vorc’ added to 0.2 ml of human serum or tissue extract and the mixture incubated at room temp-ature for I 11 to ensure binding of the iron to protein7. Such mixturrs \vcw subjected to starch gel electroplioresis R in Shandon tanks accomodated in a rcfrigvrator at 3’. Six samples were examined at a time by the use of large starch gel block5 IO x 18 x I.25 cm, prep-cd using a borate buffer 0.02; JZ strength and pH ().oj. For a continuous buffer systctn the coniplctiieIitar?. bridge buffer was 0.3 ,lI with ;L pH of 8.6. IXoading of the gels Lvitli samples was achivlwl by nioistcninji (utilizing capillary attraction) 1.25 cm scluarcs of \1Xatrnan No. 3 filtct- ppcr and inserting them into slits prwiouslv cut in the gel along the line of application. ;1 constant current of 20 11~1 \vas applied for 17 11 to effect optimum migration of tlte proteins. Following elwtrophorcsis, gels were cut horizontally into three sliccg. ‘flit uppr surface of the lowrr third was stained with nigrosin to dcmonstratc the distribution of the protein fractions. The bottom surface of the upper third was stained for fcrritin using Pcrle’s stain (2?,, potassium ferroc\.anide in z”,, hydrochloric acid). ‘lllv niidtllc third wts cut longitudinally with a dcrmatome blade into six :;‘t~ips of cclual \vidtll so as to isolate each electrophoretic pattern, the s;ampl~~application slits being uwtl as the guides for determining the actual positi 9iling of each cut. \\.‘itliout separating tllcgtxl the whole slice \vas cut tra:;:;;ia-sely at 2-mm intcwals, across its width in par;L11vl to the line of sample application. The six cle~tropllorctic patterns derived from one gel Lucre therclq. divided into an equal number of fractions, each corresponding in positioning and approximate size to its companion fractions in otlwr t~lectrophoreti(~ strips. Prior to c,ounting tlic ratlioactivit\. of each gel fraction, wc~11 ~vas n+$icd Iwfore Iwing placed in a labelled countingL bottle. ‘fhc wt~iglit of t~acli slices \vas uwd ;I’_;;L means of cAculating radioacti\-itv per mm of gel strip (we 1~~10~). ‘Ilit> ratlioactivit>- contained in c~cli gel fraction was determined using a Paimx wll-t!pv scintillation counter. (‘aunts \vcre made for a standard tinit, of IOO WC. I%wkgrountl vomits were in the order of 400, The specimtn counts corrected for I~ackground ranged bctucen zero and 5000. I
IRON

BINDING

BY TISSUE

PROTEINS

373

’ b y =1I_x_ w’ 1 Where :

Y = counts/roe

set/mm of each gel fraction Y’ = total counts/Ioo set in each fraction IN’ = weight of each fraction b 1

= sum of weights of all the gel fractions = length of each strip in mm.

in each strip

The counts/roe set/mm of gel was plotted against the distance of migration. 3. Immunotitration. A modification of the method of GelllO was used. Serial (geometric) dilutions of a standard serum of known total iron binding capacity (TIBC) and of the tissue extracts were made. Using a barbiturate buffer, 19; agar gels 3-mm thick were prepared on 8 x 8 cm glass plates. Four sets, each of five wells 4 mm in diameter were cut with a cork borer. The distance separating the outer wells from the central wells was 5 mm. For future orientation, a small hole was cut in the upper left hand corner of the gel. Specific anti-transferrin sera were placed in the four central wells of each plate. Dilutions I : I, I : 2, I :4, I : 16, I: 32, I : 64 and I : 128 of the standard serum or tissue extract with normal saline were placed in the peripheral wells. The maximum dilution at which precipitin bands could be identified was noted at 24 h. A comparison of the lowest dilution of tissue extract producing a precipitin band with that of the standard serum gave an approximate evaluation of the concentration of transferrin in the extract. Knowing the concentration of protein in each sample, these figures were used to calculate the amount of transferrin per gram of protein for each tissue. RESULTS

Starch gel electrophoresis Fig. I shows the typical protein patterns derived from brain, kidney, muscle, liver and spleen when subjected to electrophoresis in a single starch block. The separation

Fig. I. Electrophoretic patterns of proteins obtained in a single starch gel block and stained with nigrosin.

from

serum

Clin.

and tissue

Chirn.

Acta,

extracts

18 (1967)

when

run

371-376

achieved indicates that the electrophorctic technique is suitable for making comparative studies of the migrations of different 591’e-accepting proteins derived from various tissues. Fig. z relates the tissue and serum protein patterns and secondly,

the presence

or not of stainable

ferritin.

to firstl!., All patterns

5g1;e radioactivity exhibit

an early

Fig. L. Showing the relation of (a) the 5”F~ radioactivitv scan to (b) protein patterns and (c) stainable ferritin. It is sho\vn how differentiation of fcrritin (A) from transfcrrin (JS)can lx matlc. The arrows mark the point of application of the cxtrncts. (The y-globulin bands migrating towards the anode arc not included as they had no 591:e-bintling propertics.)

zone of radioactivity on the cathode side of the specimen application slit. A similar effect was also observed when pure human albumin was subjected to electrophoresis after incubation with 5YFeCI,. W:hen serum was examined, the main peak of 591;e radioactivity corresponded to the P-globulin

band, the position

known to be occupied by transferrin7.

A similar

result was obtained when kidney extract was studied, suggesting that the 5eI;eaccepting protein in kidney is transferrin. In contrast, a peak of radioactivity corresponding in position to a band staining for ferritin was demonstrable in brain, liver and spleen. Of the five varieties of tissue examined only muscle failed to give a peak of radioactivity due to the presence of either transferrin or ferritin. The tissue specificity of the above electrophoretic and radioactive characteristics was investigated by examining tissues removed from several cadavers. Fig. 3 gives details of the migration of 5QFe-accepting tissue proteins derived from brain (x 7),kidney ( x 4), liver (x 5) and spleen (x 6).

Fig. 4 shows the concentrations of transferrin (expressed as a percentage of that contained in a standard serum with a total iron-binding capacity of 345 pg per IOO ml) found in brain (x3), kidney (x6), liver (x5), muscle ( x 5) and spleen (x 5). Of the five varieties of tissues investigated only kidney and liver contained significant amounts of transferrin. This protein was present in the highest titre in the kidney Clin. Chim. Acta,18 (1967)

371-376

IRON BINDING BY TISSUE PROTEINS

0.L

375

TISSUE

Fig. 3. Showing the distance migrated by 58Fe when added to serum and tissue extract. The serum peak of 59Fe radioactivity was considered to represent a migration of IOO~/. Line A represents the distance migrated by ferritin and line B represents that of transferrin (cf. A and B in Fig. 2). Fig. 4. Showing the concentration of transferrin per gram of protein in various tissues expressed as a percentage of the concentration found in a standard serum.

extracts. In this group, however, the range of scatter was considerable The liver extracts contained concentrations with a range of 3 to 8%.

(5 to 32%).

DISCUSSION

This investigation confirms previous observations that ferritin is present in liver12 and spleen13. By using a starch gel electrophoretic technique, it has been possible ;o differentiate qualitatively between the 5gFe uptake of ferritin and transferrin. Transferrin was detected in the electrophoretic patterns made from kidney protein extracts, but could not be detected in those made from extracts of brain, liver, muscle and spleen. The presence or not of transferrin in serum and tissues demonstrated by starch gel electrophoresis was confirmed when an immunotitration technique was used in brain”,

the study on brain, kidney, muscle and spleen. In contrast, the immunotitration revealed in liver the presence of appreciable amounts of transferrin though no transferrin peak could be demonstrated on starch gel electrophoresis. This discrepancy Another may be due to ferritin having a greater avidity for 58Fe than transferrin. possibility is that liver transferrin, though immunologically similar, is not electrophoretically identical to that in serum and other tissues. It might be argued that the presence of appreciable amounts of transferrin in kidney and liver could be the results of the contamination of the tissue homogenates with serum trapped in the blood vessels of the intact tissues. In order to avoid this contamination, during the preparation of each extract the fragmented tissue was thoroughly washed prior to homogenisation. This technique was considered sufficient to remove all but trace amounts of serum. Confirmation of this assumption was obtained by the demonstration of no 59Fe-accepting proteins in muscle extracts. characKopp et al. 13, and Theron et aLIz, in their studies on the electrophoretic teristics of ferritin, were able to demonstrate three distinct bands. That with the Clin. Chim.

Acta,

18

(1967) 371-376

most rapid migration contained go”,, of the total frrritin, the next thv last contained I”,,. These workers considered that the slowr duwd by aggregations of fcrritin molecul~5. In the present study \vas demonstrated. This descrepancv betwcn earl& observations abl\. due to the fact that these workers subjected a concentrated

contained (I”~, and bands \vcrc proonI>. a single band and our5 is proI)clxtract of fcrritin

to electrophoresis,

to the intact intra-

n-hcxreas wc studied the relationship

of ferritin

ctllular

protein pattern. In the radio-isotopic scans of all the preparations including serum tlicw \vas an early zone of increased radioactivity. This phenomenon is considerctl to IX the result of clrtachment of 5uI;c loosel~~ bound to albumin during the process of elcctrophoresis. That such mechanism occurs, is suggested I)>, the Lvork of Cairns-SmithL1 in his stud> of the binding of dves to albumin. Corroboration of the application of this finding to of this earl!, the loose binding bf b!‘I;e to albumin was given bv the demonstration trailing peak when clectrophoresis

was carried out using a mixture

of S81;e and human

albumin. The demonstration

of significant concentrations of transferrin in liver extracts is in accord with the observations madt by Iirarndstrup I, Mandell and Asofsk>r and ThorbcckeS, previously referred to in the introduction to this paper, and lends support to the view that liver cells manufacture transfcrrin. Of particular interest is the observation that transferrin is also contained in significant quantities in renal tissue. This would suggest that cellular elements in kidney are capable of manufacturing or storing transfrrrin. The low titre of serum transfcrrin found in chronic renal diwaw is usually explained by the existence of a permeabilit~~ defect in the glomrruli allowing loss of transferrin into the urine. It is possible that a reduced serum transferrin 1~~1 under these circumstances is, in part, due to a failure of the kidnev to produce transferrin.

\I’c, would like to thank Professor Stank!. Alstead for his support and cnwuragemrnt during the preparation of this paper, and also aknowlcdgc gratefully tht technical assistance provided by Mr. Stephen Miller.