Lipid binding specificity of bovine α-lactalbumin: A multidimensional approach

Many soluble proteins are known to interact with membranes in partially disordered states, and the mechanism and relevance of such interactions in cellular processes are beginning to be understood. Bovine α-lactalbumin (BLA) represents an excellent prototype for monitoring membrane interaction due to its conformational plasticity. In this work, we comprehensively monitored the interaction of apo-BLA with zwitterionic and negatively charged membranes utilizing a variety of approaches. We show that BLA preferentially binds to negatively charged membranes at acidic pH with higher binding affinity. This is supported by spectral changes observed with a potential-sensitive membrane probe and fluorescence anisotropy measurements of a hydrophobic probe. Our results show that BLA exhibits a molten globule conformation when bound to negatively charged membranes. We further show, using the parallax approach, that BLA penetrates the interior of negatively charged membranes, and tryptophan residues are localized at the membrane interface. Red edge excitation shift (REES) measurements reveal that the immediate environment of tryptophans in membrane-bound BLA is restricted, and the restriction is dependent on membrane lipid composition. We envision that understanding the mechanism of BLA–membrane interaction would help in bioengineering of α-lactalbumin, and to address the mechanism of tumoricidal and antimicrobial activities of BLA–oleic acid complex.     

Denaturation of α-lactalbumin and myoglobin by the anionic biosurfactant rhamnolipid

Glycolipid biosurfactants (GBS) are promising environmentally friendly alternatives to chemical surfactants. Surfactants interact with proteins in many applications, often leading to significant changes in protein properties. Given GBS' marked difference in structure compared to traditional chemical surfactants, it is of interest to investigate their impact on protein structure and stability. Here we combine spectroscopic and calorimetric studies to analyze the interactions between the anionic GBS rhamnolipid (RL) and two model proteins α-lactalbumin in the Ca^2 +-free apo-form (αLA) and myoglobin (Mb), whose interactions with traditional surfactants are well known. RL denatures αLA at sub-cmc concentrations (0.1–1 mM) while Mb is only denatured above the cmc, i.e. in the presence of RL micelles. Denaturation leads to increased α-helicity, similar to the effect of SDS. The proteins bind approximately the same amount of RL by weight as SDS. However, RL employs a denaturation mechanism which combines features from non-ionic surfactants (very slow unfolding kinetics and few unfolding steps) with those of SDS (unfolding below the cmc in the case of αLA and the ability to unfold stable proteins in the case of Mb). We ascribe these features to RL's weakly acidic carboxylic head group and complex hydrophobic tail, which lead to a low cmc and low protein affinity. These features restrict the concentration range where RL monomers can bind and denature proteins while still allowing micelles to bind and denature to a significant extent.     

Cytotoxicity of bovine α-lactalbumin: Oleic acid complexes correlates with the disruption of lipid membranes

HAMLET/BAMLET (Human/Bovine α-Lactalbumin Made Lethal to Tumors) is a tumoricidal substance composed of partially unfolded human/bovine α-lactalbumin (HLA/BLA) and several oleic acid (OA) molecules. The HAMLET mechanism of interaction involves an insufficiently understood effect on the membrane or its embedded components. We examined the effect of BLAOA (bovine α-lactalbumin complexed with oleic acid, a HAMLET-like substance) and its individual components on cells and artificial lipid membranes using viability staining and metabolic dyes, fluorescence spectroscopy, leakage integrity assays and microscopy. Our results show a dose-dependency of OA used to prepare BLAOA on its ability to induce tumor cell death, and a correlation between leakage and cell death. BLAOA incorporates into the membrane, tightens the lipid packing and lowers their solvent accessibility. Fluorescence imaging reveals that giant unilamellar vesicles (GUVs) develop blebs and eventually collapse upon exposure to BLAOA, indicating that the lipid packing reorganization can translate into observable morphological effects. These effects are observed to be local in GUVs, and a tightly packed and solvent-shielded lipid environment is associated with leakage and GUV disruption. Furthermore, the effects of BLAOA on membrane are pH dependent, with an optimum of activity on artificial membranes near neutral pHs. While BLA alone is effective at membrane disruption at acidic pHs, OA is ineffective in a pH range of 4.5 to 9.1. Taken together, this supports a model where the lipid, fatty acid and protein components enhance each other's ability to affect the overall integrity of the membrane.     

Iron-dependent binding of bovine milk α-casein with holo-lactoferrin, but not holo-transferrin

Bovine milk α-casein has been identified as an iron- and heme-binding protein. However, the physiological role of its iron-binding remains to be elucidated in more detail. α-Casein was immobilized on CNBr-activated Sepharose 4B beads, and the α-casein agarose beads efficiently bound hemin as well as ferrous ammonium sulfate (Fe(2+)) as compared with control beads. Additionally, α-casein-beads bound bovine holo-lactoferrin (Lf), but not holo-transferrin. Lf caused the release of Fe(2+) which had bound to the α-casein-agarose beads beforehand. These results suggest that bovine α-casein iron-dependently binds holo-bovine Lf more strongly than Fe(2+), and that strong binding between them may play a physiological role in regulating iron homeostasis in the bovine mammary gland.     

Structure and binding ability of self-assembled α-lactalbumin protein nanotubular gels

Partial hydrolysis of whey-based α-lactalbumin (α-La) with Bacillus licheniformis protease (BLP) induces the formation of nanotubular structures in the presence of calcium ions by a self-assembly process. α-La nanotubes (α-LaNTs) exist in the form of regular hollow strands with well-defined average dimensions. The growth of nanotubes induces the formation of stiff transparent protein gels due to the well-arranged networks that the strands can form; these gels can be used for entrapment, transportation, and target delivery of bioactive agents in the industry. High purity of α-La (free of other whey protein fractions) is desirable for nanotube formation; however, pure proteins are very expensive and not practically obtained for industrial applications. Thus, the purpose of this research was to construct α-LaNTs from an α-La preparation with lower purity and to study the gelation phenomena triggered by the self-assembled nanotubes. Some structural features of nanotube gels and their active agent-binding abilities were also investigated. A lower amount of α-LaNTs was observed when low purity α-La was used for nanotube formation. Nanotube growth induced gel formation and higher gel stiffness was obtained when compared to α-La hydrolysates. α-La was denatured after hydrolysis and self-assembly, and remarkable changes were observed in the α-helix and β-sheet domains of α-La structure. Increased intensity in Amide I and II regions indicated potential locations for binding of active agents to α-LaNTs. Whey-based α-La without much purification can be used to produce nanotubular gels and these gels can be considered carrying matrices for active agents in various industrial applications.     

Interaction of human α-lactalbumin with fatty acids: Determination of binding parameters

The interaction of holo- and apo-forms of human alpha-lactalbumin with fatty acids was studied by a partition equilibrium method. Apo-alpha-lactalbumin, obtained by treatment with EDTA, displays one binding site for fatty acids, the association constants for oleic and palmitic acids being 1.9.10(6) and 4.2.10(5) M(-1), respectively. However, holo-alpha-lactalbumin was unable to bind fatty acids as measured by this technique. Likewise, no fatty acids bound to holo-alpha-lactalbumin, isolated using nondenaturing conditions, were detected by gas chromatography. These results demonstrate that the conformational change induced in alpha-lactalbumin by the removal of calcium enables the protein to interact with fatty acids.     

Conformational stability of LYLA1, a synthetic chimera of human lysozyme and bovine α-lactalbumin

LYLA1 is a chimeric protein mainly consisting of residues originating from human lysozyme but in which the central part (Ca²⁺-binding site and helix C) of bovine α-lactalbumin has been inserted. The equilibrium unfolding of this hybrid protein has been examined by circular dichroism and tryptophan fluorescence techniques. The reversible denaturation process induced by temperature or by addition of chemical denaturant is three-state in the case of apo-LYLA1 and two-state in the presence of Ca²⁺. The Ca²⁺-bound form of the chimera exhibits higher stability than both wild-type lysozyme and α-lactalbumin. The stability of the apo-form, however, is intermediate between that of the parent molecules. Unfolding of apo-LYLA1 involves an intermediate state that becomes populated to a different extent under various experimental conditions. Combination of circular dichroism with bis-ANS fluorescence experiments has permitted us to characterize the acid state of LYLA1 as a molten globule. Furthermore our results strongly suggest the presence of multiple denatured states depending on external conditions. Received: 24 April 1996 / Accepted: 4 September 1996     

Comparison of the enthalpy state of vesicles of different size by their interaction with α-lactalbumin

The characteristics of small unilamellar, large unilamellar and large multilamellar vesicles of dimyristoylphosphatidylcholine and their interaction with α-lactalbumin are compared at pH 4. (1) By differential scanning calorimetry and from steady-state fluorescence anisotropy data of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene it is shown that the transition characteristics of the phospholipids in the large unilamellar vesicles resemble more those of the multilamellar vesicles than of the small unilamellar vesicles. (2) The size and composition of the lipid-protein complex formed with α-lactalbumin around the transition temperature of the lipid are independent of the vesicle type used. Fluorescence anisotropy data indicate that in this complex the motions of the lipid molecules are strongly restricted in the presence of α-lactalbumin. (3) The previous data and a comparison of the enthalpy changes, $\text{ΔH}$, of the interaction of the three vesicle types with α-lactalbumin allow us to derive that the enthalpy state of the small unilamellar vesicles just below 24°C is about 24 kJ/mol lipid higher than the enthalpy state of both large vesicle types at the same temperature. The abrupt transition from endothermic to exothermic $\text{ΔH}$ values around 24°C for large vesicles approximates the transition enthalpy of the pure phospholipid     

Compact state of a protein molecule with pronounced small-scale mobility: bovine α-lactalbumin

We describe a novel physical state of a protein molecule which is nearly as compact as the native state and has pronounced secondary structure, but differs from the native state by the large increase of thermal fluctuations (in particular, by the large mobility of side groups). This state has been characterized in detail for the acid form of bovine -lactalbumin as a result of the study of physical properties of this state by a large variety of different methods (hydrodynamics, diffuse X-ray scattering, circular dichroism and infrared spectra, polarization of the luminescence, proton magnetic resonance, deuterium exchange and microcalorimetry). It has been shown that bovine -lactalbumin can be transformed into a similar state by thermal denaturation. This process is thermodynamically two state (i.e. all-or-none transition), which means that this state differs from the native one by a phase transition of the first order.Abbreviations B-LA bovine -lactalbumin - Gu·HCl guanidine hydrochloride - CD circular dichroism - UV ultraviolet - IR infra-red - NMR nuclear magnetic resonance. Differen forms of B-LA are abbreviated as follows - N native form - A acid form - T temperature-denatured form - U unfolded form (by 6 M Gu·HCl or 8 M urea). All forms have intact S-S bonds     

Effect of pH and Glucose on the Stability of α-lactalbumin

The objective of this study is to understand the influence of pH and effect of cosolvent (glucose) on the stabilization of bovine α-lactalbumin by using ultrasonic techniques. Values of density, ultrasonic velocity and viscosity were measured for bovine α-lactalbumin (5 mg/ml) dissolved in phosphate buffer (pH 2, 5, 7, 9 and 12) solutions mixed with and without the cosolvent at 30 °C. These measurements were used to calculate few thermo-acoustical parameters such as adiabatic compressibility, intermolecular free length, acoustic impedance, relaxation time, relative association constant, the partial apparent specific volume and the partial apparent specific adiabatic compressibility for the said systems. The obtained results revealed a strong comparison between the effects of acidic and alkaline pH values on protein denaturation, i.e., the acidic pH are instantaneous and are of less magnitude whereas alkaline pH are slower but sharper. Further the present study supports the fact that the presence of glucose stabilizes α-lactalbumin against denaturation due to pH variation, which may be due to the strengthening of non-covalent interactions and the steric exclusion effect.     

The transition from the native to the acid-state characterized by multi-spectroscopy approach: Study for the holo-form of bovine α-lactalbumin

The transition of the holo-form of bovine α-lactalbumin from the native (N) to the pH-generated acidic-state (A-state) was analyzed by probing its tertiary and secondary structure using a concerted spectroscopic approach combining near- and far-UV circular dichroism (CD), electrospray ionization ion mobility mass spectrometry (ESI-IM-MS), vibrational circular dichroism (VCD), and Fourier transform infrared spectroscopy (FTIR) in the attenuated total reflection (ATR) and transmission (TR) modes. The spectroscopic results, which relied on the interaction of an electromagnetic field with different molecular targets, confirmed the decay of extensive rigid side-chain packing interactions during the pH-induced N → A-state transition and revealed the targets' dependence on secondary structural changes. Independent analyses of the spectral changes using two methods of multivariate analysis, such as principal component analysis and two-dimensional correlation spectroscopy, revealed small but significant differences in the secondary structure as a result of the all-or-none transition. The cooperativity of the transition was quantitatively described using values corresponding to the mid-point (t_m) and width of the transition (Δt_m). The averages of the two parameters, calculated using the data collected by the different probes, were equal to 3.5 ± 0.2 and 0.6 ± 0.1(SE), respectively. The variable two-state nature of the cooperative N → A-state transition confirmed that the protonation of the side chain carboxyl groups on the Asp and Glu residues and that the release of a Ca^2 + ion induced structural changes on both the secondary and tertiary levels. The changes have been confirmed by results obtained from the concerted spectroscopic approach.     

Interaction of Curcumin and Diacetylcurcumin with the Lipocalin Member β-Lactoglobulin

The binding of curcumin (CUR) and diacetylcurcumin (DAC) to bovine beta-lactoglobulin (BLG) genetic variant B was investigated by fluorescence and circular dichroism techniques. The binding parameters including number of substantive binding sites and the binding constants have been evaluated by fluorescence quenching method. The distance (r) between donor (BLG) and acceptor (CUR and DAC) was obtained according to the Förster’s theory of non-radiative energy transfer. The far-UV circular dichroism spectra were used to investigate the possible changes in the secondary structure of BLG in the presence of CUR and DAC and showed that these two ligands change the α-helix and random coil contents of this protein to some extent. The visible circular dichroism spectra indicated that the optical activity during the ligand binding was observed due to the induced-protein chirality. All of the achieved results suggested the important role of the phenolic OH group of CUR in the binding process resulted in more affinity of CUR than DAC for binding to BLG.     

Investigating the binding interactions of galantamine with β-amyloid peptide

The anti-Alzheimer’s agent galantamine is known to possess anti-amyloid properties. However the exact mechanisms are not clear. We studied the binding interactions of galantamine with amyloid peptide dimer (Aβ_1–40) through molecular docking and molecular dynamics simulations. Galantamine’s binding site within the amyloid peptide dimer was identified by docking experiments and the most stable complex was analyzed by molecular dynamics simulation. These studies show that galantamine was interacting with the central region of the amyloid dimer (Lys16–Ala21) and the C-terminal region (Ile31–Val36) with minimum structural drift of Cα atom in those regions. Strikingly, a significant drift was observed at the turn region from Asp23-Gly29 (Cα atom RMSD = 9.2 Å and 11.6 Å at 50 fs and 100 fs respectively). Furthermore, galantamine’s binding mode disrupts the key pi–pi stacking interaction between aromatic rings of Phe19 (chain A) and Phe19 (chain B) and intermolecular hydrogen bonds seen in unbound peptide dimer. Noticeably, the azepine tertiary nitrogen of galantamine was in close proximity to backbone CO of Leu34 (distance <3.5 Å) to stabilize the dimer conformation. In summary, the results indicate that galantamine binding to amyloid peptide dimer leads to a significant conformational change at the turn region (Asp23–Gly29) that disrupts interactions between individual β-strands and promotes a nontoxic conformation of Aβ_1–40 to prevent the formation of neurotoxic oligomers.     

A comparison of the crosslinking abilities of glutaraldehyde,formaldehyde and α-hydroxyadipaldehyde with bovine serum albumin and casein

Summary The crosslinking effects of formaldehyde, -hydroxyadipaldehyde and glutaraldehyde have been compared by various techniques. Using a micro-Ouchterlony technique with an aldehyde treated bovine serum albumin-rabbit anti-bovine serum albumin system it was found that glutaraldehyde prevented precipitin line formation except at very high titres of antibody. The effects of formaldehyde and -hydroxyadipaldehyde were less marked. Cellulose acetate electrophoresis of aldehyde treated bovine serum albumin showed an increase in mobility compared with the untreated protein. Starch gel electrophoresis of aldehyde fixed liver slices showed no protein loss after glutaraldehyde fixation whereas the other aldehydes permitted proteins to be extracted. Polyacrylamide gel electrophoresis of the aldehyde treated bovine serum albumin showed a little change in mobility after formaldehyde and -hydroxyadipaldehyde treatment and a little polymer formation. Glutaraldehyde on the other hand produced much polymer. These findings were confirmed by gel filtration with Sephadex G-200. Intermolecular crosslinking with glutaraldehyde was dependant on the aldehyde concentration.     

Organization and dynamics of tryptophans in the molten globule state of bovine α-lactalbumin utilizing wavelength-selective fluorescence approach: Comparisons with native and denatured states

Bovine α-lactalbumin (BLA) is known to be present in molten globule form in its apo-state (i.e., Ca²⁺ depleted state). We explored the organization and dynamics of the functionally important tryptophan residues of BLA in native, molten globule and denatured states utilizing the wavelength-selective fluorescence approach. We observed red edge excitation shift (REES) of 7 nm for the tryptophans in native BLA. Interestingly, we show here that BLA tryptophans exhibit considerable REES (8 nm) in its molten globule state. Taken together, these results indicate that tryptophan residues in BLA in native as well as molten globule states experience motionally restricted environment. We further show that even the denatured form of BLA exhibits a modest REES of 3 nm, indicating that the tryptophans are shielded from bulk solvent, even when denatured, due to the presence of residual structure around tryptophan(s). This is further supported by wavelength-dependent changes in fluorescence anisotropy and lifetime for BLA tryptophans. These novel results constitute one of the first reports of REES in the molten globule state of proteins, and could provide vital insight into the role of tryptophans in the function of BLA in its molten globule state in particular, and other partially ordered proteins in general.     

Characterization of the extracellular,water-insoluble α-D-glucans of oral streptococci by methylation analysis,and by enzymic synthesis and degradation

Methylation analysis of water-insoluble α-D-glucans synthesized from sucrose by culture filtrates from several strains of Streptococcus spp. has proved that all of the glucans were highly branched and that the chains contained (1→6)- and (1→3)-linked D-glucose residues not involved in branch points. Hydrolysis of the glucans with a specific endo-(1→3)-α-D-glucanase demonstrated that the majority of the (1→3)-linked glucose residues were arranged in sequences. D-Glucose was the major product of the hydrolysis, and a small proportion of nigerose was also released. The use of a specific endo-(1→6)-α-D-glucanase similarly indicated that the glucans also contained sequences of (1→6)-linked α-D-glucose residues, and that those chains were branched. Two D-glucosyltransferases (GTF-S and GTF-I), which reacted with sucrose to synthesize a soluble glucan and a water-insoluble glucan, respectively, were separated from culture filtrates of S. mutans OMZ176. The soluble glucan was characterized as a branched (1→6)-α-D-glucan, whereas the insoluble one was a relatively linear (1→3)-α-D-glucan. The hypothesis is advanced that the glucosyltransferases can transfer glucan sequences by means of acceptor reactions similar to those proposed by Robyt for dextransucrase, leading to the synthesis of a highly branched glucan containing both types of chain. The resulting structure is consistent with the evidence obtained from methylation analysis and enzymic degradations, and explains the synergy displayed when the two D-glucosyltransferases interact with sucrose. Variations in one basic structure can account for the characteristics of water-insoluble glucans from S. sanguis and S. salivarius, and for the strain-dependent diversity of S. mutans glucans.     

Characterization of an alternative low energy fold for bovine α-lactalbumin formed by disulfide bond shuffling

Bovine α-lactalbumin (αLA) forms a misfolded disulfide bond shuffled isomer, X-αLA. This X-αLA isomer contains two native disulfide bridges (Cys 6-Cys 120 and Cys 28-Cys 111) and two non-native disulfide bridges (Cys 61-Cys 73 and Cys 77-Cys 91). MD simulations have been used to characterize the X-αLA isomer and its formation via disulfide bond shuffling and to compare it with the native fold of αLA. In the simulations of the X-αLA isomer the structure of the α-domain of native αLA is largely retained in agreement with experimental data. However, there are significant rearrangements in the β-domain, including the loss of the native β-sheet and calcium binding site. Interestingly, the energies of X-αLA and native αLA in simulations in the absence of calcium are closely similar. Thus, the X-αLA isomer represents a different low energy fold for the protein. Calcium binding to native αLA is shown to help preserve the structure of the β-domain of the protein limiting possibilities for disulfide bond shuffling. Hence, binding calcium plays an important role in both maintaining the native structure of αLA and providing a mechanism for distinguishing between folded and misfolded species.     

Temporal and spatial expression of biologically active human factor VIII in the milk of transgenic mice driven by mammary-specific bovine α-lactalbumin regulation sequences

Hemophilia A is one of the major inherited bleeding disorders caused by a deficiency or abnormality in coagulation factor VIII (FVIII). Hemophiliacs have been treated with whole plasma or purified FVIII concentrates. The risk of transmitting blood-borne viruses and the cost of highly purified FVIII are the major factors that restrict prophylaxis in hemophilia therapy. One of the challenges created by the biotechnology revolution is the development of methods for the economical production of highly purified proteins in large scales. Recent developments indicate that manipulating milk composition using transgenesis has focused mainly on the mammary gland as a bioreactor to produce pharmaceuticals. In the present study, a hybrid gene containing bovine -lactalbumin and human FVIII cDNA was constructed for microinjection into the pronuclei of newly fertilized mouse eggs. The LA-hFVIII hybrid gene was confirmed to be successfully integrated and stably germ-line transmitted in 12 (seven females/five males) lines. Western-blot analysis of milk samples obtained from eight of the transgenic founders and F1 offspring indicated that the recombinant hFVIII was secreted into the milk of the transgenic mice. The concentrations of rFVIII ranged from 7.0 to 50.2g/ml, over 35–200-fold higher than that in normal human plasma. Up to 13.4U/ml of rFVIII was detected in an assay in which rFVIII restored normal clotting activity to FVIII-deficient human plasma.     

Investigation of γE-crystallin target protein binding to bovine lens alpha-crystallin by small-angle neutron scattering

α-Crystallin, one of the main constituent proteins in the crystalline lens, is an important molecular chaperone both within and outside the lens. Presently, the structural relationship between α-crystallin and its target proteins during chaperone action is poorly understood. It has been hypothesised that target proteins bind within a central cavity. Small-angle neutron-scattering (SANS) experiments in conjunction with isotopic substitution were undertaken to investigate the interaction of a target lens protein (γE-crystallin) with α-crystallin (α_H) and to measure the radius of gyration (Rg) of the proteins and their binary complexes in solution under thermal stress. The size of the α_H in D₂O incubated at 65 °C increased from 69 ± 3 to 81 ± 5 Å over 40 min, in good agreement with previously published small-angle X-ray scattering (SAXS) and SANS measurements. Deuterated γE-crystallin in H₂O buffer (γE_D/H₂O) and hydrogenous γE-crystallin in D₂O buffer (γE_H/D₂O) free in solution were of insufficient size and/or too dilute to provide any measurable scattering over the angular range used, which was selected primarily to investigate γE:α_H complexes. The evolution of the aggregation size/shape as an indicator of α_H chaperone action was monitored by recording the neutron scattering in different H:D solvent contrasts under thermally stressed conditions (65 °C) for binary mixtures of α_H, γE_H, and γE_D. It was found that Rg(α_H:γE_D/D₂O) > Rg(α_H:γE_H/D₂O) > Rg(α_H/D₂O) and that Rg(α_H:γE_H/D₂O) ≈ Rg(α_H/D₂O). The relative sizes observed for the complexes weighted by the respective scattering powers of the various components imply that γE-crystallin binds in a central cavity of the α-crystallin oligomer, during chaperone action.     

Determination of the operational molarity of solutions of bovine α-chymotrypsin, trypsin, thrombin and factor Xa by spectrofluorimetric titration

Several esters of 4-methylumbelliferone and 2-naphthol were synthesized and examined as possible spectrofluorimetric titrants for bovine alpha-chymotrypsin, trypsin, thrombin, Factor Xa and for subtilisin Novo. 4-Methylumbelliferyl p-guanidinobenzoate hydrochloride (MUGB) is a satisfactory titrant for alpha- and beta-trypsin, thrombin and Factor Xa and 4-methylumbelliferyl p-(NNN-trimethylammonium)cinnamate (MUTMAC) is a good titrant for alpha-chymotrypsin. The amount of enzyme used for spectrofluorimetric titration is 0.02-3.00nmol and the amount of 4-methylumbelliferone liberated is independent of the concentration of titrant and stoicheiometrically equal to the amount of enzyme used. Results obtained with MUGB and MUTMAC have been checked by spectrophotometric titration with p'-nitrophenyl p-guanidinobenzoate hydrochloride and p-nitrophenyl N(2)-acetyl-N(1)-benzylcarbazate respectively. p-Nitrophenyl N(2)-acetyl-N(1)-(9-anthrylmethyl)carbazate has been synthesized; it did not react with alpha-chymotrypsin. A satisfactory spectrofluorimetric titrant for subtilisin Novo was not discovered.