691:(pI) is the pH of a solution at which the net primary charge of a protein becomes zero. At a solution pH that is above the pI the surface of the protein is predominantly negatively charged and therefore like-charged molecules will exhibit repulsive forces. Likewise, at a solution pH that is below the pI, the surface of the protein is predominantly positively charged and repulsion between proteins occurs. However, at the pI the negative and positive charges cancel, repulsive electrostatic forces are reduced and the attraction forces predominate. The attraction forces will cause aggregation and precipitation. The pI of most proteins is in the pH range of 4–6. Mineral acids, such as
84:
increasing concentration of co-ions. The presence of these solvation layers cause the protein to have fewer ionic interactions with other proteins and decreases the likelihood of aggregation. Repulsive electrostatic forces also form when proteins are dissolved in water. Water forms a solvation layer around the hydrophilic surface residues of a protein. Water establishes a concentration gradient around the protein, with the highest concentration at the protein surface. This water network has a damping effect on the attractive forces between proteins.
158:. This phase occurs at a slower rate. During the final step, called aging in a shear field, the precipitate particles repeatedly collide and stick, then break apart, until a stable mean particle size is reached, which is dependent upon individual proteins. The mechanical strength of the protein particles correlates with the product of the mean shear rate and the aging time, which is known as the Camp number. Aging helps particles withstand the fluid shear forces encountered in pumps and centrifuge feed zones without reducing in size.
91:
122:. For example, basic residues on a protein can have electrostatic interactions with acidic residues on another protein. However, solvation by ions in an electrolytic solution or water will decrease protein–protein attractive forces. Therefore, to precipitate or induce accumulation of proteins, the hydration layer around the protein should be reduced. The purpose of the added reagents in protein precipitation is to reduce the hydration layer.
851:, are frequently used to precipitate proteins because they have low flammability and are less likely to denature biomaterials than isoelectric precipitation. These polymers in solution attract water molecules away from the solvation layer around the protein. This increases the protein–protein interactions and enhances precipitation. For the specific case of polyethylene glycol, precipitation can be modeled by the equation:
672:
129:
103:
178:, compresses the solvation layer and increases protein–protein interactions. As the salt concentration of a solution is increased, the charges on the surface of the protein interact with the salt, not the water, thereby exposing hydrophobic patches on the protein surface and causing the protein to fall out of solution (aggregate and precipitate).
298:
protein aggregation and precipitation. Chaotropes or "water structure breakers," have the opposite effect of
Kosmotropes. These salts promote an increase in the solvation layer around a protein. The effectiveness of the kosmotropic salts in precipitating proteins follows the order of the Hofmeister series:
724:
to a solution may cause proteins in the solution to precipitate. The solvation layer around the protein will decrease as the organic solvent progressively displaces water from the protein surface and binds it in hydration layers around the organic solvent molecules. With smaller hydration layers, the
66:
content on the surface have low solubility in an aqueous solvent. Charged and polar surface residues interact with ionic groups in the solvent and increase the solubility of a protein. Knowledge of a protein's amino acid composition will aid in determining an ideal precipitation solvent and methods.
144:
Protein precipitate formation occurs in a stepwise process. First, a precipitating agent is added and the solution is steadily mixed. Mixing causes the precipitant and protein to collide. Enough mixing time is required for molecules to diffuse across the fluid eddies. Next, proteins undergo a
1032:
depend on the pH of the solution. Anionic polyelectrolytes are used at pH values less than the isoelectric point. Cationic polyelectrolytes are at pH values above the pI. It is important to note that an excess of polyelectrolytes will cause the precipitate to dissolve back into the solution. An
297:
Kosmotropes or "water structure stabilizers" are salts which promote the dissipation / dispersion of water from the solvation layer around a protein. Hydrophobic patches are then exposed on the protein's surface, and they interact with hydrophobic patches on other proteins. These salts enhance
83:
migrate towards charged surface residues on the protein, forming a rigid matrix of counterions on the protein's surface. Next to this layer is another solvation layer that is less rigid and, as one moves away from the protein surface, contains a decreasing concentration of counterions and an
703:
caused by the mineral acids. For this reason isoelectric point precipitation is most often used to precipitate contaminant proteins, rather than the target protein. The precipitation of casein during cheesemaking, or during production of sodium caseinate, is an isoelectric precipitation.
1093:
as they move though the tubes of the reactor. Turbulent flow is promoted through wire mesh inserts in the tube. The tubular reactor does not require moving mechanical parts and is inexpensive to build. However, the reactor can become impractically long if the particles aggregate slowly.
1075:
Batch reactors are the simplest type of precipitation reactor. The precipitating agent is slowly added to the protein solution under mixing. The aggregating protein particles tend to be compact and regular in shape. Since the particles are exposed to a wide range of
283:= Absolute temperature. When water molecules in the rigid solvation layer are brought back into the bulk phase through interactions with the added salt, their greater freedom of movement causes a significant increase in their entropy. Thus, Δ
635:
149:
phase, where submicroscopic sized protein aggregates, or particles, are generated. Growth of these particles is under
Brownian diffusion control. Once the particles reach a critical size (0.1 ÎĽm to 10 ÎĽm for high and low
398:
1088:
In tubular reactors, feed protein solution and the precipitating reagent are contacted in a zone of efficient mixing then fed into long tubes where precipitation takes place. The fluid in volume elements approach
656:
is the salt concentration. The ideal salt for protein precipitation is most effective for a particular amino acid composition, inexpensive, non-buffering, and non-polluting. The most commonly used salt is
467:
62:
amino acid residues on the protein's surface. Hydrophobic residues predominantly occur in the globular protein core, but some exist in patches on the surface. Proteins that have high hydrophobic
262:
989:
805:
190:
when the right concentration of the salt is reached in solution. The hydrophobic patches on the protein surface generate highly ordered water shells. This results in a small decrease in
1130:
909:
661:. There is a low variation in salting out over temperatures 0 °C to 30 °C. Protein precipitates left in the salt solution can remain stable for years-protected from
528:
725:
proteins can aggregate by attractive electrostatic and dipole forces. Important parameters to consider are temperature, which should be less than 0 °C to avoid
481:
solubility curve of the type shown. The relationship between the solubility of a protein and increasing ionic strength of the solution can be represented by the
553:
79:
solution. These repulsive forces between proteins prevent aggregation and facilitate dissolution. Upon dissolution in an electrolyte solution, solvent
31:
industry protein precipitation is used to eliminate contaminants commonly contained in blood. The underlying mechanism of precipitation is to alter the
154:
fields, respectively), by diffusive addition of individual protein molecules to it, they continue to grow by colliding into each other and sticking or
1155:
Zellner; et al. (June 2005). "Quantitative validation of different protein precipitation methods in proteome analysis of blood platelets".
304:
1102:
1134:
406:
1061:
There are numerous industrial scaled reactors than can be used to precipitate large amounts of proteins, such as recombinant
726:
700:
829:
for plasma protein fractionation relies on solvent precipitation with ethanol to isolate individual plasma proteins.
90:
832:
a clinical application for the use of methanol as a protein precipitating agent is in the estimation of bilirubin.
220:
1157:
931:
743:
207:
1028:
and polyphosphates can form extended networks between protein molecules in solution. The effectiveness of these
699:
are used as precipitants. The greatest disadvantage to isoelectric point precipitation is the irreversible
671:
1230:
1006:
128:
32:
102:
20:
857:
478:
24:
1109:
with a continuous flow of reactants and products in a well-mixed tank. Fresh protein feed contacts
848:
730:
491:
187:
998:
1174:
734:
688:
291:
1033:
example of polyelectrolyte flocculation is the removal of protein cloud from beer wort using
174:
is the most common method used to precipitate a protein. Addition of a neutral salt, such as
1166:
1034:
1029:
658:
175:
1066:
206:
of the ordered water molecules relative to the molecules in the bulk solution. The overall
1062:
630:{\displaystyle I={\begin{matrix}{\frac {1}{2}}\end{matrix}}\sum _{i=1}^{n}c_{i}z_{i}^{2}}
1224:
1046:
737:
the relationship between the dielectric constant and protein solubility is given by:
696:
151:
118:
Dispersive or attractive forces exist between proteins through permanent and induced
28:
1106:
1080:
for a long period of time, they tend to be compact, dense and mechanically stable.
1077:
826:
729:, pH and protein concentration in solution. Miscible organic solvents decrease the
692:
155:
1025:
662:
171:
76:
59:
55:
548:
is the ionic strength of the solution, which is attributed to the added salt.
146:
80:
63:
51:
36:
733:
of water, which in effect allows two proteins to come close together. At the
1113:
that already contains precipitate particles and the precipitation reagents.
1090:
1178:
1170:
393:{\displaystyle \mathrm {PO_{4}^{3-}>SO_{4}^{2-}>COO^{-}>Cl^{-}} }
1045:
Metal salts can be used at low concentrations to precipitate enzymes and
1021:
844:
840:
721:
713:
191:
1131:"Protein Precipitation Plates and Tubes - Pharmaceutical International"
717:
199:
119:
75:
Repulsive electrostatic forces form when proteins are dissolved in an
1110:
825:
is a constant that relates to the dielectric constant of water. The
1050:
482:
54:
of proteins in aqueous buffers depends on the distribution of
665:
and bacterial contamination by the high salt concentrations.
214:, of the process is given by the Gibbs free energy equation:
23:
of biological products in order to concentrate proteins and
287:
becomes negative and precipitation occurs spontaneously.
1215:
Bioseparations: downstream processing for biotechnology.
462:{\displaystyle \mathrm {NH_{4}^{+}>K^{+}>Na^{+}} }
564:
934:
860:
746:
556:
494:
409:
307:
223:
27:
them from various contaminants. For example, in the
35:of the solvent, more specifically, by lowering the
983:
903:
799:
629:
522:
461:
392:
256:
925:is a protein–polymer interaction coefficient and
1203:. John Wiley & Sons, Inc. New York, NY 2001.
1196:(2nd Edition). Prentice Hall International. 2001
1217:John Wiley & Sons, Inc. New York, NY 1988.
1210:John Wiley & Sons, Inc. New York, NY 1994.
921:is a protein–protein interaction coefficient,
821:is the dielectric constant of the mixture and
477:The decrease in protein solubility follows a
8:
1024:, carboxymethylcellulose, polyacrylic acid,
257:{\displaystyle \Delta G=\Delta H-T\Delta S.}
1189:Oxford University Press. New York, NY 2003.
984:{\displaystyle x=(\mu _{i}-\mu _{i}^{0})RT}
800:{\displaystyle \log S=k/e^{2}+\log S^{0}\,}
966:
961:
948:
933:
900:
859:
796:
790:
771:
762:
745:
621:
616:
606:
596:
585:
567:
563:
555:
519:
493:
452:
436:
423:
418:
410:
408:
383:
367:
345:
340:
321:
316:
308:
306:
222:
39:of the solute by addition of a reagent.
1187:Bioseparations Science and Engineering.
1122:
1098:Continuous stirred tank reactors (CSTR)
667:
275:= Enthalpy change upon precipitation, Δ
124:
86:
1194:Bioprocess Engineering: Basic Concepts
1053:frequently used are Ca, Mg, Mn or Fe.
279:= Entropy change upon precipitation,
7:
708:Precipitation with miscible solvents
647:is the ion charge of the salt and
449:
445:
433:
415:
411:
380:
376:
364:
360:
357:
337:
333:
313:
309:
245:
233:
224:
182:Energetics involved in salting out
14:
1049:from solutions. Polyvalent metal
1017:Flocculation by polyelectrolytes
904:{\displaystyle \ln(S)+pS=X-aC\,}
670:
544:is a salt-specific constant and
127:
101:
89:
42:Biochemical laboratory technique
972:
941:
917:is the polymer concentration,
873:
867:
836:Non-ionic hydrophilic polymers
114:Attractive electrostatic force
1:
1013:is the absolute temperature.
536:= solubility of the protein,
523:{\displaystyle \log S=B-KI\,}
71:Repulsive electrostatic force
813:is an extrapolated value of
198:, and a larger decrease in
1247:
1201:Bioseparations Engineering
683:Isoelectric precipitation
540:is idealized solubility,
1041:Polyvalent metallic ions
1208:Bioprocess Engineering.
473:Salting out in practice
271:= Free energy change, Δ
1171:10.1002/elps.200410262
1057:Precipitation reactors
1007:universal gas constant
985:
905:
801:
631:
601:
524:
463:
394:
258:
986:
906:
802:
632:
581:
525:
464:
395:
259:
140:Precipitate formation
96:Ionic solvation layer
21:downstream processing
17:Protein precipitation
932:
858:
849:polyethylene glycols
744:
554:
492:
469:least precipitation
407:
400:least precipitation
305:
221:
971:
731:dielectric constant
626:
428:
403:Most precipitation
353:
329:
301:Most precipitation
188:spontaneous process
33:solvation potential
1137:on 18 October 2006
999:chemical potential
981:
957:
901:
797:
627:
612:
579:
520:
459:
414:
390:
336:
312:
254:
46:General principles
19:is widely used in
1213:Belter, Paul A.
1185:Harrison et al.,
735:isoelectric point
716:solvents such as
689:isoelectric point
575:
292:Hofmeister series
186:Salting out is a
1238:
1182:
1147:
1146:
1144:
1142:
1133:. Archived from
1127:
1105:reactors run at
1084:Tubular reactors
1065:from a solution.
1030:polyelectrolytes
1001:of component I,
990:
988:
987:
982:
970:
965:
953:
952:
910:
908:
907:
902:
806:
804:
803:
798:
795:
794:
776:
775:
766:
677:Solubility curve
674:
659:ammonium sulfate
636:
634:
633:
628:
625:
620:
611:
610:
600:
595:
580:
576:
568:
529:
527:
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387:
372:
371:
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328:
320:
263:
261:
260:
255:
176:ammonium sulfate
131:
105:
93:
1246:
1245:
1241:
1240:
1239:
1237:
1236:
1235:
1221:
1220:
1192:Shuler et al.,
1158:Electrophoresis
1154:
1151:
1150:
1140:
1138:
1129:
1128:
1124:
1119:
1100:
1086:
1073:
1063:DNA polymerases
1059:
1043:
1019:
944:
930:
929:
856:
855:
838:
786:
767:
742:
741:
710:
685:
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655:
646:
602:
578:
577:
552:
551:
490:
489:
475:
448:
432:
405:
404:
379:
363:
303:
302:
295:
219:
218:
184:
169:
164:
142:
135:
134:Hydration layer
132:
116:
109:
108:Hydration layer
106:
97:
94:
73:
48:
43:
12:
11:
5:
1244:
1242:
1234:
1233:
1223:
1222:
1219:
1218:
1211:
1204:
1197:
1190:
1183:
1165:(12): 2481–9.
1149:
1148:
1121:
1120:
1118:
1115:
1099:
1096:
1085:
1082:
1078:shear stresses
1072:
1071:Batch reactors
1069:
1058:
1055:
1042:
1039:
1018:
1015:
992:
991:
980:
977:
974:
969:
964:
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947:
943:
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72:
69:
47:
44:
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13:
10:
9:
6:
4:
3:
2:
1243:
1232:
1231:Biotechnology
1229:
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1226:
1216:
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1209:
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1202:
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1188:
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1123:
1116:
1114:
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1108:
1104:
1097:
1095:
1092:
1083:
1081:
1079:
1070:
1068:
1067:
1064:
1056:
1054:
1052:
1048:
1047:nucleic acids
1040:
1038:
1036:
1031:
1027:
1023:
1016:
1014:
1012:
1008:
1004:
1000:
996:
978:
975:
967:
962:
958:
954:
949:
945:
938:
935:
928:
927:
926:
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920:
916:
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891:
888:
885:
882:
879:
876:
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864:
861:
854:
853:
852:
850:
846:
842:
835:
833:
830:
828:
824:
820:
816:
812:
791:
787:
783:
780:
777:
772:
768:
763:
759:
756:
753:
750:
747:
740:
739:
738:
736:
732:
728:
723:
719:
715:
707:
705:
702:
698:
697:sulfuric acid
694:
690:
682:
673:
668:
666:
664:
660:
654:
650:
645:
641:
637:
622:
617:
613:
607:
603:
597:
592:
589:
586:
582:
572:
569:
560:
557:
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535:
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513:
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501:
498:
495:
488:
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486:
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472:
470:
453:
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429:
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401:
384:
373:
368:
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341:
330:
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317:
299:
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290:
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217:
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209:
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197:
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148:
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125:
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104:
99:
92:
87:
85:
82:
78:
70:
68:
65:
61:
57:
53:
45:
40:
38:
34:
30:
29:biotechnology
26:
22:
18:
1214:
1207:
1200:
1193:
1186:
1162:
1156:
1139:. Retrieved
1135:the original
1125:
1107:steady state
1101:
1087:
1074:
1060:
1044:
1020:
1010:
1002:
994:
993:
922:
918:
914:
913:
839:
831:
827:Cohn process
822:
818:
814:
810:
809:
727:denaturation
712:Addition of
711:
701:denaturation
693:hydrochloric
686:
652:
648:
643:
639:
638:
550:
545:
541:
537:
533:
532:
476:
402:
300:
296:
284:
280:
276:
272:
268:
266:
211:
203:
195:
185:
170:
156:flocculating
143:
117:
74:
49:
16:
15:
1141:14 December
1026:tannic acid
663:proteolysis
208:free energy
172:Salting out
167:Salting out
81:counterions
77:electrolyte
60:hydrophobic
56:hydrophilic
1206:Lydersen.
1117:References
1035:Irish moss
843:, such as
485:equation:
479:normalized
147:nucleation
64:amino acid
52:solubility
37:solubility
1199:Ladisch.
1091:plug flow
959:μ
955:−
946:μ
892:−
865:
784:
751:
583:∑
511:−
499:
385:−
369:−
350:−
326:−
246:Δ
240:−
234:Δ
225:Δ
210:change, Δ
1225:Category
1179:15895463
1022:Alginate
845:dextrans
841:Polymers
722:methanol
714:miscible
192:enthalpy
1005:is the
997:is the
718:ethanol
200:entropy
162:Methods
120:dipoles
1177:
1111:slurry
25:purify
152:shear
1175:PMID
1143:2006
1103:CSTR
1051:ions
1009:and
847:and
695:and
687:The
483:Cohn
443:>
430:>
374:>
355:>
331:>
58:and
50:The
1167:doi
781:log
748:log
720:or
496:log
202:, Δ
194:, Δ
1227::
1173:.
1163:26
1161:.
1037:.
862:ln
817:,
204:S,
1181:.
1169::
1145:.
1011:T
1003:R
995:ÎĽ
979:T
976:R
973:)
968:0
963:i
950:i
942:(
939:=
936:x
923:a
919:P
915:C
898:C
895:a
889:X
886:=
883:S
880:p
877:+
874:)
871:S
868:(
823:k
819:e
815:S
811:S
792:0
788:S
778:+
773:2
769:e
764:/
760:k
757:=
754:S
653:i
649:c
644:i
640:z
623:2
618:i
614:z
608:i
604:c
598:n
593:1
590:=
587:i
573:2
570:1
561:=
558:I
546:I
542:K
538:B
534:S
517:I
514:K
508:B
505:=
502:S
454:+
450:a
446:N
438:+
434:K
425:+
420:4
416:H
412:N
381:l
377:C
365:O
361:O
358:C
347:2
342:4
338:O
334:S
323:3
318:4
314:O
310:P
285:G
281:T
277:S
273:H
269:G
267:Δ
252:.
249:S
243:T
237:H
231:=
228:G
212:G
196:H
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