1. Emoglobine SinteticheEmoglobine Sintetiche
Andrea MozzarelliAndrea Mozzarelli
andrea.mozzarelli@unipr.itandrea.mozzarelli@unipr.it
Dipartimento di FarmaciaDipartimento di Farmacia
Università di ParmaUniversità di Parma
Parma, ItalyParma, Italy
Anemo 2015, 6 marzo., IRCCS Policlinico San Donato
2. • Perché sono necessarie
• Quali sono
• Perché non sono ancora disponbili
4. Problemi associati alle trasfusioni
• Necessità di accurate tipizzazioni;
• Limitazioni nel numero di donatori sani;
• Possibili contaminazioni da agenti infettivi;
• Alti costi delle analisi per escludere contaminazioni virali;
• Patologie indotte da trasfusioni, per. es. in neonati pre-
termine, quali retinopatie e malattie polmonari croniche;
• Ridotto tempo di utilizzo del sangue intero (42 giorni);
• Variazione delle proprietà del sangue dal tempo di
conservazione.
5. Variazione delle proprietà dei globuli rossi durante la conservazione
Bennet-Guerrero et al. (2007) PNAS 104, 17063
6. Esigenze cliniche non soddisfatte
dalle trasfusioni
• Trattamento di eventi emorragici severi (incidenti d’auto, in
montagna, sui campi di battaglia) in luoghi lontani da ospedali;
• Apporto di ossigeno a tessuti ischemici non raggiungibili dai
globuli rossi;
• Trattamento di pazienti immuno-reattivi a tutti i tipi di sangue;
• Trattamento di pazienti che rifiutano il sangue;
• Trattamento di bambini nati pre-termine;
• Carenza di sangue nei paesi del Terzo Mondo e dell’Est Europeo.
7. Quali sono le alternative al sangue?
• Globuli rossi da cellule staminali
• Produzione di globuli rossi del Gruppo 0,
donatore universale
• Animali transgenici (maiali e mucche)
• Piante OGM (tabacco)
• Fluorocarburi
• Emoglobine sintetiche
11. Il tetramero di emoglobina, libero nel
plasma, dissocia in dimeri che hanno
alta affinità per l’ossigeno
+
12. I dimeri di emoglobina vanno incontro a
ultrafiltrazione renale
Nefrotossicità
Patologie emolitiche e da trasfusioni multiple
13. Altri eventi associati alla presenza di dimeri e tetrameri
di emoglobina libera nel plasma
NO scavenging
vasocostrizione
Extravasazione Hb
dimeri
Scavanging NO
Vasocostrizione, ipertensione
Aggregazione piastrinica e coagulazione (???)
Stress ossidativi
Formazione di
radicali liberi
autossidazione
14. Modificazioni chimiche e approccci biotecnologici
per:
prevenire la dissociazione del tetramero
aumentare le dimensioni della molecola
ridurre la reattività con NO
Biochimica Biotecnologia
x
x
x
x
x
Purificazione
emoglobina
Modificazioni
biochimiche Espressione
eterologa
18. Tuttavia, i vari derivati dell’emoglobina
sono etereogenei strutturalmente e
funzionalmente, e sono presenti stress
ossidativi
l’indice terapeutico, il rapporto
benefici/rischi …
21. Un sostituto del sangue su base emoglobinica è un farmaco
“difficile” da realizzare:
Per le elevate quantità da iniettare perché sia efficace come trasportatore di
ossigeno (centinaia di grammi, effetti osmotici)
Per le possibili impurezze e eterogeneità dei prodotti ottenuti per
modificazioni chimiche
Per i molteplici meccanismi associati all’emoglobina libera nel plasma
Un approccio integrato biochimico, fisiologico e farmacologico, utilizzando
biomarkers plasmatici e biomarkers di organi potrà portare a sviluppare
Emoglobine Sintetiche con alti indici beneficio/rischio.
CONCLUSIONICONCLUSIONI
24. RBC Free Zone Plasma
RBC
NO
VesselWall
Unstirred Layer
BloodFlow
RBC Free Zone Plasma
RBC
Unstirred Layer
NO
HOOH
ONOO
-
Hb
EC EC
Cell-free Hb and The Vasculature
(Alayash, A., Nature Rev. Drug Disc. 3:152-159, 2004)
NO scavenging
Increase in Blood Pressure & Blood Flow
25. From subunits to tetramer: a probability distributionFrom subunits to tetramer: a probability distribution
PEG/tetramer
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
fractionontotalprotein
0.00
0.05
0.10
0.15
0.20
30. Biphasic behaviour of PEG-HbBiphasic behaviour of PEG-Hbdeoxydeoxy
: a global analysis: a global analysis
na=1.33+0.09
p50a=0.80+0.12 torr
p50b=3.18+0.08 torr
nb=2.54+0.19
a
b
PEGPEG--HbHbdeoxydeoxy
and PEG-Hband PEG-Hboxyoxy
at low concentration show similar oxygen binding propertiesat low concentration show similar oxygen binding properties
25 µM
165 µM
550 µM
11 µM
a, b
PEG-Hboxy
(p50=0.85+0.02 torr, n=1.34+0.04)
Caccia et al. Bioconjugate Chem., 20 (2009) 1356-1366
31. NEM/PEGHb: hemoglobin PEGylated in anaerobic conditions, 6.7 NEM,NEM/PEGHb: hemoglobin PEGylated in anaerobic conditions, 6.7 NEM, ~~7.7 PEG 5K7.7 PEG 5K
NPM/PEGHb: hemoglobin PEGylated in anaerobic conditions, 6.6 NPM,NPM/PEGHb: hemoglobin PEGylated in anaerobic conditions, 6.6 NPM, ~~7.5 PEG 5K7.5 PEG 5K
Sample affinity/cooperativity
(pH 7.4, 37°C)
Borh effect
(∆p50/ ∆pH)
p50 (torr) n
HbA 17.74±0.25 2.99±0.18 -4.98
NEM/PEG-Hb 9.73±0.31 1.61±0.10 -1.55
NPM/PEG-Hb 14.98±0.56 1.48±0.10 -3.84
PEG-Hboxy
6.79±0.21 1.52±0.07 -1.25
Portörö et al. BBA 1784 (2008) 1402-1409
NPM/PEGHb preserves an hemoglobin-like affinity and alkaline Bohr effectNPM/PEGHb preserves an hemoglobin-like affinity and alkaline Bohr effect
Effect of protein charges modification on functional propertiesEffect of protein charges modification on functional properties
32. CO binding after rapid mixingCO binding after rapid mixing
T R
PEGylation perturbes T to R transitionPEGylation perturbes T to R transition
Caccia et al. Bioconjugate Chem., 20 (2009) 1356-1366
33. R
T
CO rebinding after flash photolysis:CO rebinding after flash photolysis: detection of quaternary statesdetection of quaternary states
time (sec)
1e-7 1e-6 1e-5 1e-4 1e-3 1e-2
N(t)
0.0
0.2
0.4
0.6
0.8
1.0
HbA0
PEG-Hboxy
PEG-Hb
deoxy
dimersdimers
PEGylation slows down R to TPEGylation slows down R to T
transition and dimers reassociationtransition and dimers reassociation
Caccia et al. Bioconjugate Chem., 20 (2009) 1356-1366
tetramerstetramers
34. time (sec)
1e-7 1e-6 1e-5 1e-4 1e-3 1e-2
N(t)
0.0
0.2
0.4
0.6
0.8
1.0
98 uM
13 uM
PEG-HbPEG-Hbdeoxydeoxy
log τ
-8 -6 -4 -2 0
g(logτ)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
log τ
-3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0
g(logτ)
0.00
0.05
0.10
0.15
0.20
0.25
MEMMEM
Concentration dependence of kinetic rebinding to CO: diluitionConcentration dependence of kinetic rebinding to CO: diluition
produces theproduces the disappearance of T state redinding phasedisappearance of T state redinding phase
35. PEG-HbPEG-Hboxyoxy
time (sec)
1e-7 1e-6 1e-5 1e-4 1e-3 1e-2
N(t)
0.0
0.2
0.4
0.6
0.8
1.0 MEMMEM
Concentration dependence of kinetic rebinding to CO: diluitionConcentration dependence of kinetic rebinding to CO: diluition
produces theproduces the disappearance of T state redinding phasedisappearance of T state redinding phase
36. O2
100%
O2
20%
O2
5%
O2
1%
CO2He
POR1 POR2 PORT3
DIL POR1 POR2 POR3 POR4
OUTPUT
Microspectrophotometer
(humidified)
Environics 200Environics 4000 OUTPUT
Spectrophotometer
Microspectrophotometer
(not humidified)
Thermostatted humidifier
Cuvette
Equilibration chamber
humidifier
Oxygen binding measurements: instrumental setOxygen binding measurements: instrumental set
upup
Fractional saturation with oxygen and fractional
concentration of oxidized hemes have been determined by
fitting each individual spectrum to a linear combination of
deoxy, oxy, and oxidized hemoglobin spectra (Rivetti et al.
1993; Bettati and Mozzarelli 1997), recorded in solution.
This procedure provides a more precise determination of
the binding curve with respect to single wavelength
measurements.
Oxygen binding measurements – All measurements have been made in 100 mM HEPES,
1 mM EDTA, pH 7.0, unless otherwise stated.
Experiments have been carried out using a mixing chamber connected with a cuvette
and collecting absorption spectra in the range 450-700 nm with a Varian Cary 400 Scan
spectrophotometer. The gas mixing apparatus is based on two consecutive gas mixture
generators (Environics, series 200 and 4000) to obtain a wide range of oxygen
pressures.
39. X Data
-2 0 2 4 6 8 10 12 14 16 18
YData
0
10
20
30
40
50
60
Probability assessment of a simulated tetramer hexaPEGylationProbability assessment of a simulated tetramer hexaPEGylation
40. PortPortörö et al. BBA 1784 (2008) 1402-1409örö et al. BBA 1784 (2008) 1402-1409
Positive charge neutralisation through NPM instead of NEM causedPositive charge neutralisation through NPM instead of NEM caused
less Bohr effect disappearance after PEGylationless Bohr effect disappearance after PEGylation
41. Campione Stripped dil Stripped conc Cloruri CO2
MI-13 p50= 1.047± 0.03
n= 1.27± 0.05
A=0.44
Na=1.33
p50a=0.88
nb=2.54
p50b=3.18
p50=2.027±0.04
n=1.334± 0.06
p50=2.216±0.04
n=1.383± 0.05
MI-17 p50=0.641±0.03
n=1.134± 0.07
p50=0.86±0.02
n=1.409±0.05
p50=1.40 ±0.03
n=1.41±0.06
p50=1.215±0.05
n=1.210±0.09
[heme] na nb p50a p50b Fraction a
MI-13
- Global fitting
for several
concentrations
1.33±0.09 2.54±0.19 0.80±0.12 3.18±0.08
CO2
283 uM 2.21±0.03 3.71±0.03 0.44±0.05
Cl- 110 uM 2.23±0.06 3.66±0.05 0.50±0.01
MI-17
-
CO2 3.33 ±1.45 2.63 ± 1.01
Cl- 2.24±1.82 2.50 ± 0.65
Plasmatic allosteric effectors:Plasmatic allosteric effectors: COCO22 and chlorideand chloride
47. Example: HemospanTM
(MP4), aerobically PEGylated Hb
Hemospan is a blood substitute prepared from outdated human
blood hemoglobin and maleimide polyethylene glycol. The
PEGylation is carried out with hemoglobin in the oxy form.
Hemospan has a higher oxygen affinity (p50 5 torr) and lower
cooperativity (Hill n = 1.1) than other blood substitutes currently on
clinical trials.
A similar modification carried out under anaerobic conditions in
the presence of inositol hexaphosphate leads to a PEG conjugate
with lower oxygen affinity (p50 14 torr) and higher cooperativity (Hill n
= 1.4) (anaerobically PEGylated hemoglobin = Euro-PEGHb).
48. PEGylation occurs in two steps and takes advantage of the
reactivity of lysine side chains
The extent of PEGylation can be modulated
Cysteine residues can also be PEGylated
The reactivity of specific lysine and cysteine residues depends on
the quaternary state (→ differences between Hemospan (MP4) and
anaerobically PEGylated Hb). The latter procedure protects allosteric
sites.
49.
50. S
NH2
2-iminothiolane O
O
N+ + NH
+
C
NH2
+
C
NH2
+
O-PEG
O
O
O
N NH
Thiolated Protein
Mal-Phe-PEG
PEGylated Protein
SH
S
O-PEG
O
NH2
Protein
(IT)
NH
Protein
NH
Protein
51. Val 1
K 7
K 11
K 56
K 16
α2
α1
Adapted from PDB
52. K 8 β
K 144 β
K 17 β
β2
β1
K 66 β
K 59 β
Adapted from PDB
53. High HDI Medium HDI Low HDI
Rifornimento di sangue e
Indice di sviluppo umano (HDI)Indice di sviluppo umano (HDI)
Medio HDI (n=88)Medio HDI (n=88)
29.4 milioni29.4 milioni
(36%)(36%)
Basso HDI (n=36)Basso HDI (n=36)
2.3 milioni2.3 milioni
(3%)(3%)
Alto HDI (n=54)Alto HDI (n=54)
49.4 milioni49.4 milioni
(61%)(61%)
Raccolta totale annuale: 81 milioni (178 paesi)
Source: WHO GDBS, courtesy Dr. N. Dhingra
54. % Donazioni % Populazione
%
Populazione Globale e Rifornimento diPopulazione Globale e Rifornimento di
SangueSangue
AltoMedioBasso
61
18
71
36
11
3
Source: WHO GDBS, courtesy Dr. N. Dhingra
56. Effetti avversi associati allo scavenging
dell’NO
Vasocostrizione
Ipertensione
Aggregazione piastrinica e coagulazione (?)
57. Effetti avversi associati all’ossidazione
del ferro dell’eme
Danni mediati da ROS e processi infiammatori
58. Rapporto di donazioni per 1,000 abitanti nel mondo
Donazioni per 1,000
0 - 10
10.1 - 20
20.1 - 30
30.1 - 40
40.1 - 90
No data
Sullivan P. Developing an administrative plan for transfusion medicine – a global perspective. Transfusion 2005;45:224s-240s
59. High HDI Medium HDI Low HDI
Global Blood Supply
&
Human Development Index (HDI)Human Development Index (HDI)
Medium HDI (n=88)Medium HDI (n=88)
29.4 millions29.4 millions
(36%)(36%)
Low HDI (n=36)Low HDI (n=36)
2.3 millions2.3 millions
(3%)(3%)
High HDI (n=54)High HDI (n=54)
49.4 millions49.4 millions
(61%)(61%)
Total Annual Blood Collection: 81 millions (178 Countries)
Source: WHO GDBS, courtesy Dr. N. Dhingra
60. ~ 6,000 pazienti trasfusione-dipendenti
180,000 – 240,000 unità / anno
Utilizzo di RBC per trasfusioni in pazienti
con emoglobinopatie congenite in Italia
62. “Genomics and Blood Substitutes in the 21st
Century
Europe – EuroBloodSubstitutes”
Progetto FP6, 2004-2007
“Approcci biotecnologici alla terapia delle patologie da
ipoossigenazione: emoglobine modificate
Progetto Fondazione Cariparma, 2008-2011
Euro-PEG-Hb
64. Coniugazione con polietilene glicole
(pesi molecolari da 500 a 20000)
HO-(CHOH-CHOH)n-OH
Le proteine PEGilate non sono immunogeniche
Le proteine PEGilate non sono proteolizzate
65. L’emoglobina coniugata con 6-8 PEG ha un
peso molecolare di circa 90000 e un volume
idrodinamico doppio di quello
dell’emoglobina.
67. Biphasic behaviour of PEG-HbBiphasic behaviour of PEG-Hbdeoxydeoxy
: a global analysis: a global analysis
na=1.33+0.09
p50a=0.80+0.12 torr
p50b=3.18+0.08 torr
nb=2.54+0.19
a
b
PEGPEG--HbHbdeoxydeoxy
and PEG-Hband PEG-Hboxyoxy
at low concentration show similar oxygen binding propertiesat low concentration show similar oxygen binding properties
25 µM
165 µM
550 µM
11 µM
a, b
PEG-Hboxy
(p50=0.85+0.02 torr, n=1.34+0.04)
Caccia et al. Bioconjugate Chem., 20 (2009) 1356-1366
69. Effetti associati allo scavenging dell’NO?
- HBOC presentano un’attività NITRITO REDUTTASICA
con produzione di NO a basse pressioni di ossigeno
(Gladwin et al. 2005)
- PEG-HBOC presentano una aumentata attività
NITRITO REDUTTASICA (Kluger et al., 2008, 2009)
Effetti associati a ROS e RNS ?
Editor's Notes
Rouhani FJ, Dor FJMF, Cooper DKC. Investigation of red blood cells from alpha 1, 3-galactosyltransferase-knockout pigs for human blood transfusion. Transfusion 2004;44:1004-12.
This is why we set up an experimental platform . This platform forsees classical bioclinical assays on guinea pigs, where a correction for the hemoglobin intereference is considered, will be coupled to a proteomic investigation on guinea pigs heart, liver and pancreas.
This high-throughput and large scale analysis allows to identify and quantify at molecular level the response of the protein machinery to the transfusion of HBOCs, highlighting the mechanisms and the markers of therapeutic and potentially lethal adverse effects.
This procedure translates into a clinically relevant model of acute arterial hemorrhage, followed by restoration of lost volume and oxygen carrying capacity when HBOC is transfused.
This scheme represents our understanding of the interplay between cell-free Hb and the vasculature with special focus on the role of nitric oxide.
NO produced by endothelial cells lining the vessel wall can diffuse into a large area in its short half life (2-3 sec) (Yellow). It crosses several physical barriers; RBC free zone (created because of intravascular flow of RBCs), unstirred layer of plasma around RBCs and The RBC’s membrane. Whatever little NO diffuses into RBCs and react with Hb can however, be controlled by RBCs antioxidative enzymes.
However, in the case of cell free Hb; this Hb can reach the vessel wall (unlike RBCs) react avidly with NO, resulting in vasoconstriction, hypertension. More important the consequences of the depletion of NO result in an oxidative environment resulting in damaging effect on Hb and the vessel wall.
SO, IDEALY YOU WOULD WANT TO HAVE A CELL-FREE HB “BLOOD SUBSTITUTES” THAT REACT LESS WITH NO WITH SATBLE HEME AGAINST OXIDATIVE ATTACKES BY OXIDANTS.
Jack Lancaster: based some of his mathematical calculations on Our animal studies that we reported few years ago. We found that DCLHb (Tetramer) and polymerized versions induced similar hypertensive responses ..We incidentally just published a paper in Analt. Chemistry to show that this is form of polymerization lacks any tetramer in solution
A densitometric analysis on protein bands separated by SDS PAGE has allowed to quantify relative fraction of differently PEGylated globin chains in these derivatives. A comparison between two different PEGylated hemoglobin, with 3 and 6 average PEG molecules per tetramer, quantify what already observed on the gel, that is the distribution shifts towards, higher PEGylated subunits. But, what about the tetramer?
Assuming that pegylation takes place on each subunit with the same probability regardless its level of PEGylation, we calculated the probability of having tetramers with one, two, t… PEG molecules.
On Hb-PEGdeoxy, this probability distribution was calculated, ranging from 0 to 16. and an average weight number of 6.6 has been obtained, in excellent agreement with the direct biochemical evaluation. Even considering the information loss due to electrophoretic separation, this probability re-tetramerization suggests that PEGylation generates a complex mixture of products.
We investigated the dimensional pattern also in native conditions:
size exclusion chromatography (with Superdex HiLoad 200pg) gave us the picture of the dimensional distribution in native conditions.
Here we reported the distributions for PEG-Hboxy(red) and PEG-Hbdeoxy (light blue). These distributions are very broad and show several peaks spanning in a wide range of molecular weights. But If the SEC is carried out in deoxygenated conditions, the profiles show very large changes. Both samples, even if with a different extent, appear more homogeneous and move towards higher molecular weight simply by deoxygenation.
.
Likewise, simply by increasing the concentration of the sample, similar changes were observed: we observed more homogeneous profiles.
So, diluition and oxygenation, both dimerization promoting events for native haemoglobin, even with a different extent in these two PEGylated derivatives, promotes heterogeneity. The most simple explanation is that Pegylation destabilizes th tetramer, causing dimerization and dimensional pattern broadening.
Giving the fact that, as demonstrated by SDS PAGE, subunits with different PEGylation degrees are present, tetramers with the same total PEG number can be composed by dimers with different PEGylation degre: 5+1, 4+2 or 3+3, or 6+0.
Dimensional distributions have been also characterized by native gradient electrophoresis on PEGHboxy and PEGHbdeoxy, in comparison with unmodified hemoglobin, at high concentration and in oxygenated conditions. PEGylation gives a large increase of the volume of the hemoglobin, as one can see from this native gradient gel in which the migration of the unmodified hemolgobin is compared with two PEGylated haemoglobins. Densitometric profile shows that, even if the average PEG chains are similar, different patterns are observed. Noteworthy, a few percent of unmodified, known as toxic, tetramer is still present.
Even if with SEC separation of concentrated solution showed similar profile, with this analysis, having a higher resolution, different dimensional patterns emerge, regardless their similar number of PEGylated chains per tetramer.
PEGylation is not homogeneous (0,1,2,3,4 per subunit)
PEGylation distribution is protocol dependent
The fraction of non-PEGylated tetramers is still present even in hexaPEGylated derivatives
From a functional point of view, the first characterised functional property of PEGylated hemoglobins has been, obsiously, the oxygen binding. Oxygen binding measurements have been carried out on PEG-Hboxy and PEG-Hbdeoxy, in comparison with stroma free hemoglobin. Since we observed an effect of concentration on dimensional pattern and it is known that dimers, favoured by diluition, bind oxygen with high affinity and almost no cooperativity we checked different concentrations.
In the concentration range of 100 uM, closed circles, PEG-Hbdeoxy showed a biphasic oxygen binding behaviour, which tends to become monophasic diluiting the sample to 10 uM, open circles, and to move towards an high affinity-low cooperativity curve as observed for PEG-Hboxy . The latter did not show relevant differences changing the concentration, as stroma free hemoglobin as well.
In view of the strong concentration dependence of Peg-Hbdeoxy a set of oxygen binding curves at different concentrations have been analysed by a global fit to a composition of two, independent, oxygen binding species. The fit has given that only by changing the relative population of the two species, a and b, it was possible to convolute all the concentration. the concentration dependence of these equilibrium is shown here. These results suggest that at least two, (non-interconverting oxygen binding) species are present in solution, and their equilibrium is concentration dependent. The fraction of the two species, once it has reached approximately the same value, does not change anymore. Once again, it can be supposed that PEG-Hbdeoxy tends to dimerize more easily than native hemoglobin does, and Pegylation, probably does not allow a complete re-tetramerization because a part of the high affinity form seems not participate in the dimer-tetramer equilibrium or alternatively, this fraction can correposond to a population of “frozen” R-like structures. For Peg-Hboxy, affinity and cooperativity did not show relevant changes, probably because this PEGylation protocol tends to stabilise an R-like structure, functionally almost indistinguishable from dimers. R state and dimers are very similar from a functional point of view
-PEGylated hemoglobin is heterogeneous, showing at least two species with different cooperativity and different affinity for oxygen.
The effect of protein charges modification has been also considered from a funcional point of view comparing two different pegylated deoxyhemoglobin, where in one case negative charges are added through a negative charged sulphydryl blocker (N-propionyl-maleimide), and N-ethyl maleimide has been use as a control.
We observed that the introduction of negative charges produces a product with an affinity and a bohr effect more similar to unmodified hemoglobin, potentially interesting properties for a blood substitutes.
To characterize also how PEGylation affects conformational transitions, equlibrium studies were combined with kinetic studies: the CO binding kinetic after rapid mixing with a stopped flow apparatus has been folllowed for these derivatives. Hemoglobin, is known to have an autocatalitic behaviour, due to the T to R allosteric transition, giving an accelerated kinetic.
PEGylated hemoglobin behave Differently: PEGylated oxyhb, which have the highest affinity and the lowest cooperativity, has the faster and the more homogeneoug binding kinetic, respect to PEG-Hbdeoxy, which has an intermediate affinity and cooperativity between PEG-Hboxy and unmodified hemoglobin, has also an intermediate Co binding rate. For both pegylated samples, kinetics are concentration independent: this observation, combined with the fact that these kinetics a monophasic, suggests us PEGylation causes a prturbation of T to R transition, which does not occur in the time scale of CO binding.
Un acerta fraione di tetramero, che si comporta diversamente tra i due, ci deve essere,
Other kinetic experiments have been carried out: rebinding kinetic after nanosecond laser pulse has been followed for these products.
Tipically, hemoglobin shows a nanosecond time range rebinding, named geminate rebinding, that is a fast unimolecolar process where CO binds to heme without escaping from the heme pocket. The other CO molecules that escape into the solvent, can bimolecularly rebind: to R state hemoglobin in the hundreds of microseconds,or, if R to T transition has the time to occur, to T state, in millisecond. So T state rebinding has been used as a probe of the presence of a tetramer, and inositol hexaphosphate, a T and tetramer stabilizing allosteric effector, has been used to populate in a great extent this phase
A confirmation that the slow phase is correlated to the quaternary T state has come from the flash photolysis experiments as a function of photolysis level. The R to T transition is strongly dependent on the fractional saturation, and low ligand bond breakage lowers this propensity. As a matter of fact, lowering the photolysis level causes slow phase, disappearance.
For both PEGylated samples, we observed a markedly decrease in the T state rebinding respect to unmiodified hemoglobin in the same conditions, so we can suppose that Pegylation inhbits R to T quaternary transition and tetramer formation.
Moreover, the slow bimolecular rebinding phase, marker of a T state rebinding, tends to disappear by diluiting the sample, suggesting that diluition, that causes changes in terms of dimensional distribution and more non-cooperative binding behaviour, is related to disappearence of the rebinding to T state in pegylated deoxy Hb.
Kinetics were analysed with a kinetic model independent method, returning a probability distribution versus time, where the slow phase disapearance is even more clear.
Similarly, PEGHbO2 rebinding kinetics shows a smaller slow phase rebinding to T quaternary state respect to unmodified hemoglobin, suggesting a tetramer destabilization for this derivative as well. MEM analysis gave similar results.
as in pegylated oxyHb as well.
So, from structural and functional analysis clearly emerges that,regardless the site specificity, PEGylation destabilizes the haemoglobin tetramer, slowig down or preventing dimers reassociation and Quaternary transitions, for both samples.
Differences in affinity and cooperativity have been observed at high concentration, where the fraction of Peg-Hbdeoxy tetramer shows a more hemoglobin-like behaviour than PEgHbO2.
In collaboration with the Department of Physics of our University flash photolysis experiments have been carried out, as a sensible techniqu to investigate the structural correlated functional properties of these derivatives…….
This is the set up of the instrument…..
As a part of a consortium involving Academic and industry partners, named “genomics…”, founded by European community, we started to develop a new hemoglobin based oxygen carrier….
We learned that a PEGylated hemoglobin as a blood substitute has several useful properties. It has an increased molecular size, preventing renal filtration and reducing extravasation, PEG has low antigenic activity, and chemical modification on protein residues hinders proteolysis.
But Hemoglobin is an allosteric cooperative protein, very sensitive to chemical modifications that can alter oxygen affinity, the effect of modulators and cooperativity, so we focused our attention on this chemical modification and on its effect on functional and structural properties of hemoglobin.
In particular, mainly 2 products have been characterized: PEgHboxy, prepared following the protocol published for the preparation of MP4, and a product, here named Hb-PEGdeoxy, PEGylated under anaerobic conditions and in presence of IHP. This conditions have been coincived for the protection of CysBeta 93, a key residue that affects oxygen affinity and possibly involved in NO homeostasis, and residues involved in interactions with allosteric modulators by PEGylation. This aimed to prepare a more Hemoglobin-like product.
So through a structural and functional characterization of these derivatives, we tried to answer to some questions: between them: ….
The structural analysis of our products started from SDS PAGE. As already demonstrated, SDS PAGE is able to separate Pegylated hemoglobins in several bands. The first band migrates at the same distance than unmodified globin chains. The other bands run at high molecular weigh respect to protein standard molecular weight, due to the hydration effect of PEG mojety, but through Masspec analysis of the first 3 bands we identified the bands as corresponding to 1, 2 and 3 PEG 5000 molecules bound per subunit.
The two different samples, with an average number of 3 and 6 PEG chains per tetramer, are compared: increasing the Pegylation degree, the distribution shifts towards higher PEGylated subunits.
So in this denaturing conditions, we chracterize the heterogeneigy of the Pegylated hemoglobin in terms of subnits.
In view of the strong concentration dependence of Peg-Hbdeoxy a set of oxygen binding curves at different concentrations have been analysed by a global fit to a composition of two, independent, oxygen binding species. The fit has given that only by changing the relative population of the two species, a and b, it was possible to convolute all the concentration. the concentration dependence of these equilibrium is shown here. These results suggest that at least two, (non-interconverting oxygen binding) species are present in solution, and their equilibrium is concentration dependent. The fraction of the two species, once it has reached approximately the same value, does not change anymore. Once again, it can be supposed that PEG-Hbdeoxy tends to dimerize more easily than native hemoglobin does, and Pegylation, probably does not allow a complete re-tetramerization because a part of the high affinity form seems not participate in the dimer-tetramer equilibrium or alternatively, this fraction can correposond to a population of “frozen” R-like structures. For Peg-Hboxy, affinity and cooperativity did not show relevant changes, probably because this PEGylation protocol tends to stabilise an R-like structure, functionally almost indistinguishable from dimers. R state and dimers are very similar from a functional point of view
-PEGylated hemoglobin is heterogeneous, showing at least two species with different cooperativity and different affinity for oxygen.
Since structural an functional investigations highlighted significant differences, we carreid out an evaluation of physiological parameters related to common adverse effects in blood substitutes, such as vasoactiviy and kidney damage for massive renal fitration,, looking for a correlation. We can see that after exchange transfusion, for the three products the systemic arterial pressure comes back to normal value within 1 hour, without relevant differences. No differences have been found between these derivatives in terms of escretion through the kidney. While unmodified hemoglobin is eliminated for almost 20% within 5 hours, all three PEGylated derivatives, are eliminated for less that 1 %. This small fraction could correspond to the small fraction of unmodified tetramer we have observed in in native PAGE experiments.
So, hte modulation of functional properties shown before, does not correlate wiht differnces in physiologially parameter. We asked by ourselves if these variation are not relevant or we are not studying in depth this biological issue.
